Immune perturbation likely affects the development of Kaposi sarcoma (KS) among people infected with the KS-associated herpesvirus (KSHV). We tested whether KSHV-seropositive individuals or cases of classic KS (cKS), which typically originates in the leg, had differing delayed-type hypersensitivity (DTH) in the forearm or leg.

We demonstrate that conditioning of mammalian cells by electroporation with nanosecond pulsed electric field (nsPEF) facilitates their response to the next nsPEF treatment. The experiments were designed to unambiguously separate the electroporation-induced sensitization and desensitization effects. Electroporation was achieved by bursts of 300-ns, 9 kV/cm pulses (50 Hz, n = 3-100) and quantified by propidium dye uptake within 11 min after the nsPEF exposure. We observed either sensitization to nsPEF or no change (when the conditioning was either too weak or too intense, or when the wait time after conditioning was too short). Within studied limits, conditioning never caused desensitization. With settings optimal for sensitization, the second nsPEF treatment became 2.5 times (25 °C) or even 6 times (37 °C) more effective than the same nsPEF treatment delivered without conditioning. The minimum wait time required for sensitization development was 30 s, with still longer delays increasing the effect. We show that the delayed hypersensitivity was not mediated by either cell swelling or oxidative effect of the conditioning treatment; biological mechanisms underlying the delayed electrosensitization remain to be elucidated. Optimizing nsPEF delivery protocols to induce sensitization can reduce the dose and adverse side effects of diverse medical treatments which require multiple pulse applications.

Delayed-type hypersensitivity (DTH) is caused by a broad number of drugs used in clinic, and antineoplastic drugs show an elevated proportion of DTH, which potentially affects the quality of life of patients. Despite the serious problem and the negative economic impact deriving from market withdrawal of such drugs and high hospitalization costs, nowadays, there are no standard validated methods in vitro or in vivo to evaluate the sensitizing potential of drugs in the preclinical phase. Enhanced predictions in preclinical safety evaluations are really important, and for that reason, the aim of our work is to adapt in vitro DPRA, ARE-Nrf2 luciferase KeratinoSensTM, and hCLAT assays for the study of the sensitizing potential of antineoplastic agents grouped by mechanism of action. Our results reveal that the above tests are in vitro techniques able to predict the sensitizing potential of the tested antineoplastics. Moreover, this is the first time that the inhibition of the VEGFR1 pathway has been identified as a potential trigger of DTH.

Delayed drug hypersensitivity reactions are clinically diverse reactions that vary from isolated benign skin conditions that remit quickly with no or symptomatic treatment, drug discontinuation or even continued drug treatment, to the other extreme of severe cutaneous adverse reactions (SCARs) that are associated with presumed life-long memory T-cell responses, significant acute and long-term morbidity and mortality. Diagnostic "in clinic" approaches to delayed hypersensitivity reactions have included patch testing (PT), delayed intradermal testing (IDT) and drug challenges for milder reactions. Patch and IDT are, in general, performed no sooner than 4-6 weeks after resolution of the acute reaction at the maximum non-irritating concentrations. Functional in vitro and ex vivo assays have largely remained the province of research laboratories and include lymphocyte transformation test (LTT) and cytokine release enzyme linked ImmunoSpot (ELISpot) assay, an emerging diagnostic tool which uses cytokine release, typically IFN-γ, after the patient's peripheral blood mononuclear cells are stimulated with the suspected drug(s). Genetic markers such as human leukocyte antigen have shown recent promise for both pre-prescription screening as well as pre-emptive and diagnostic testing strategies.

Palladium is frequently used in dental materials, and sometimes causes metal allergy. It has been suggested that the immune response by palladium-specific T cells may be responsible for the pathogenesis of delayed-type hypersensitivity in study of palladium allergic model mice. In the clinical setting, glucocorticoids and antihistamine drugs are commonly used for treatment of contact dermatitis. However, the precise mechanism of immune suppression in palladium allergy remains unknown. We investigated inhibition of the immune response in palladium allergic mice by administration of prednisolone as a glucocorticoid and fexofenadine hydrochloride as an antihistamine. Compared with glucocorticoids, fexofenadine hydrochloride significantly suppressed the number of T cells by interfering with the development of antigen-presenting cells from the sensitization phase. Our results suggest that antihistamine has a beneficial effect on the treatment of palladium allergy compared to glucocorticoids.

Our aim was to examine the effect of demethylnobiletin on various experimental models of delayed-type hypersensitivity (DTH) reactions and to determine its influence on the mediators and enzymes involved in these reactions.

to investigate the effects cannabidiol (CBD) on delayed-type hypersensitivity (DTH) reactions and antigen-induced T-cell cytokine expression.

Hypersensitivity (HS) reactions to sulfonamide antibiotics occur uncommonly, but with potentially severe clinical manifestations. A familial predisposition to sulfonamide HS is suspected, but robust predictive genetic risk factors have yet to be identified. Strongly linked genetic polymorphisms have been used clinically as screening tests for other HS reactions prior to administration of high-risk drugs.

Neutrophils and monocytes/macrophages (MMs) play important roles in the development of cell-mediated delayed type hypersensitivity (DTH). However, the dynamics of neutrophils and MMs during the DTH reaction and how the immunosuppressant rapamycin modulates their behavior in vivo are rarely reported. Here, we take advantage of multi-scale optical imaging techniques and a footpad DTH reaction model to non-invasively investigate the dynamic behavior and properties of immune cells from the whole field of the footpad to the cellular level. During the classic elicitation phase of the DTH reaction, both neutrophils and MMs obviously accumulated at inflammatory foci at 24 h post-challenge. Rapamycin treatment resulted in advanced neutrophil recruitment and vascular hyperpermeability at an early stage (4 h), the reduced accumulation of neutrophils (> 50% inhibition ratio) at 48 h, and the delayed involvement of MMs in inflammatory foci. The motility parameters of immune cells in the rapamycin-treated reaction at 4 h post-challenge displayed similar mean velocities, arrest durations, mean displacements, and confinements as the classic DTH reaction at 24 h. These results indicate that rapamycin treatment shortened the initial preparation stage of the DTH reaction and attenuated its intensity, which may be due to the involvement of T helper type 2 cells or regulatory T cells.

Trypanosoma equiperdum is the causative agent of dourine, a parasitic venereal disease of equids. In this work, rabbits were infected with T. equiperdum strain OVI; serological tests (complement fixation test, ELISA and immunoblotting), used for the diagnosis of dourine in horses, were applied to study rabbit humoral immune response and to characterise T. equiperdum antigen pattern recognised by antibodies from infected rabbits. Moreover a protein extract of T. equiperdum strain OVI was produced and tested in skin tests on infected rabbits to detect the cell-mediated response induced by T. equiperdum, in order to evaluate its use in the field diagnosis of dourine. Sera of infected rabbits recognized in immunoblotting Trypanosoma protein bands with molecular weight below 37 kDa, providing a serological response comparable with that already observed in dourine infected horses. Moreover the trypanosome protein extract was capable to produce in vivo delayed-type hypersensitivity (DHT Type IV) in rabbits and proved itself to be non-toxic and non-sensitizing.

Drug hypersensitivity reactions can be classified as immediate or delayed. While diagnostic options for immediate reactions are well developed and standardized, delayed reactions (in many cases type IV according to Gell and Coombs) are a challenge for allergy work-up. In recent years, some in vitro markers have been proposed and used for delayed reactions, such as contact dermatitis. Primary strategy: Avoidance is difficult to achieve, especially for COVID-19 vaccinations, when immunity against infection is extremely important. The aim of our study was to evaluate the application of in vitro delayed hypersensitivity tests in COVID-19 vaccines. Seven patients with a positive history of severe delayed drug allergy were enrolled. Vein blood was collected to stimulate cells with the tested vaccines (Comirnaty, Janssen, Spikevax) and excipients with the assessment of CD40L, CD69, IL-2, IL-4, IL-6, IL-10, IFNgamma, TNFalfa, and intracellular markers: granulysin and INFgamma. In addition, basophile activation tests, patch tests, skin prick tests, and intradermal tests were performed with the tested vaccine. Finally, the decision was made to either administer a vaccine or resign. Two out of seven patients were considered positive for drug hypersensitivity in the in vitro test according to the high vaccine stimulation index measured with CD69 (6.91 and 12.18) and CD40L (5.38 and 15.91). All patch tests, BATs, and skin tests were negative. Serum interleukin measurements were inconclusive as the impact of the vaccine itself on the immunity system was high. Intracellular markers gave uncertain results due to the lack of stimulation on the positive control. CD69 and CD40L could be reliable in vitro markers for delayed hypersensitivity to COVID-19 vaccines. Patch tests, skin tests, BATs, and serum interleukins did not confirm their usefulness in our study.

Puerarin has long been used as a traditional Chinese medicine, which possesses various physiological properties, including anti-inflammation, anti-oxidative, anti-diabetic and anti-cancer activities. The aim of the current study was to investigate the effect of puerarin on delayed-type hypersensitivity (DTH) induced by ovalbumin (OVA) in mice and explore its underlying mechanisms. The results showed that puerarin significantly attenuated DTH, resulting from a decrement in footpad swelling, reduction in inflammatory cell as well as a decline in anti-OVA IgG in serum. In the homogenized supernatant of footpad tissues, the classic Th1-cytokines, including interferon (IFN)-γ was suppressed following puerarin treatment. Furthermore, a high dose of puerarin inhibited interleukin (IL)-4 production, the classic Th2-cytokine. The concanavalin A stimulation and MTT assays indicated a suppressive effect of puerarin on Th1 response via decreasing IFN-γ production in OVA-primed lymphocytes. Detailed studies revealed that puerarin modulated the Th1/Th2 balance in DTH responses, attributing to lower T-bet/GATA binding protein-3 mRNA and protein level ratios, which led to the shift change of IFN-γ/IL-4 with puerarin treatment. These findings demonstrate that puerarin alleviated inflammation in DTH triggered by OVA application via curbing inflammatory cytokines by modulating the Th1/Th2 balance. These results suggest that puerarin may be an alternative therapeutic option for the treatment of DTH.

The skin tuberculin test (TST), an example of a delayed-type hypersensitivity (DTH) reaction, is based on measuring the extent of skin induration to mycobacterial tuberculin (PPD). Little is known about the genetic basis of TST reactivity, widely used for diagnosing TB infection. The study investigated the relationship of the single base change polymorphic variants in CD14 gene (CD14(-159C/T)) with the development of DTH to PPD in BCG-vaccinated Polish Caucasian individuals. We found persistent lack of TST reactivity in about 40% of healthy subjects despite receiving more than one dose of BCG. The TST size was negatively correlated with the number of BCG inoculations. The distribution of C/T genotype was significantly more frequent among TST-negative compared with TST-positive individuals. The concentration of serum sCD14 was positively associated with mCD14 expression, but not with the TST status or CD14(-159C/T) polymorphism. A significant increase in mCD14 expression and serum sCD14 levels was found in TB group. We hypothesize that CD14(-159C/T) polymorphic variants might be one of genetic components in the response to attenuated M. bovis BCG bacilli.

Previously, we showed that mouse delayed-type hypersensitivity (DTH) can be antigen-specifically downregulated by suppressor T cell-derived miRNA-150 carried by extracellular vesicles (EVs) that target antigen-presenting macrophages. However, the exact mechanism of the suppressive action of miRNA-150-targeted macrophages on effector T cells remained unclear, and our current studies aimed to investigate it. By employing the DTH mouse model, we showed that effector T cells were inhibited by macrophage-released EVs in a miRNA-150-dependent manner. This effect was enhanced by the pre-incubation of EVs with antigen-specific antibodies. Their specific binding to MHC class II-expressing EVs was proved in flow cytometry and ELISA-based experiments. Furthermore, by the use of nanoparticle tracking analysis and transmission electron microscopy, we found that the incubation of macrophage-released EVs with antigen-specific antibodies resulted in EVs' aggregation, which significantly enhanced their suppressive activity in vivo. Nowadays, it is increasingly evident that EVs play an exceptional role in intercellular communication and selective cargo transfer, and thus are considered promising candidates for therapeutic usage. However, EVs appear to be less effective than their parental cells. In this context, our current studies provide evidence that antigen-specific antibodies can be easily used for increasing EVs' biological activity, which has great therapeutic potential.

Exosomes are endosome-derived small membrane vesicles that are secreted by most cell types including tumor cells. Tumor-derived exosomes usually contain tumor antigens and have been used as a source of tumor antigens to stimulate anti-tumor immune responses. However, many reports also suggest that tumor-derived exosomes can facilitate tumor immune evasion through different mechanisms, most of which are antigen-independent. In the present study we used a mouse model of delayed-type hypersensitivity (DTH) and demonstrated that local administration of tumor-derived exosomes carrying the model antigen chicken ovalbumin (OVA) resulted in the suppression of DTH response in an antigen-specific manner. Analysis of exosome trafficking demonstrated that following local injection, tumor-derived exosomes were internalized by CD11c+ cells and transported to the draining LN. Exosome-mediated DTH suppression is associated with increased mRNA levels of TGF-β1 and IL-4 in the draining LN. The tumor-derived exosomes examined were also found to inhibit DC maturation. Taken together, our results suggest a role for tumor-derived exosomes in inducing tumor antigen-specific immunosuppression, possibly by modulating the function of APCs.

Cysteine-type cathepsins such as cathepsin B are involved in various steps of inflammatory processes such as antigen processing and angiogenesis. Here, we uncovered the role of cysteine-type cathepsins in the effector phase of T cell-driven cutaneous delayed-type hypersensitivity reactions (DTHR) and the implication of this role on therapeutic cathepsin B-specific inhibition. Methods: Wild-type, cathepsin B-deficient (Ctsb-/-) and cathepsin Z-deficient (Ctsz-/-) mice were sensitized with 2,4,6-trinitrochlorobenzene (TNCB) on the abdomen and challenged with TNCB on the right ear to induce acute and chronic cutaneous DTHR. The severity of cutaneous DTHR was assessed by evaluating ear swelling responses and histopathology. We performed fluorescence microscopy on tissue from inflamed ears and lymph nodes of wild-type mice, as well as on biopsies from psoriasis patients, focusing on cathepsin B expression by T cells, B cells, macrophages, dendritic cells and NK cells. Cathepsin activity was determined noninvasively by optical imaging employing protease-activated substrate-like probes. Cathepsin expression and activity were validated ex vivo by covalent active site labeling of proteases and Western blotting. Results: Noninvasive in vivo optical imaging revealed strong cysteine-type cathepsin activity in inflamed ears and draining lymph nodes in acute and chronic cutaneous DTHR. In inflamed ears and draining lymph nodes, cathepsin B was expressed by neutrophils, dendritic cells, macrophages, B, T and natural killer (NK) cells. Similar expression patterns were found in psoriatic plaques of patients. The biochemical methods confirmed active cathepsin B in tissues of mice with cutaneous DTHR. Topically applied cathepsin B inhibitors significantly reduced ear swelling in acute but not chronic DTHR. Compared with wild-type mice, Ctsb-/- mice exhibited an enhanced ear swelling response during acute DTHR despite a lack of cathepsin B expression. Cathepsin Z, a protease closely related to cathepsin B, revealed compensatory expression in inflamed ears of Ctsb-/- mice, while cathepsin B expression was reciprocally elevated in Ctsz-/- mice. Conclusion: Cathepsin B is actively involved in the effector phase of acute cutaneous DTHR. Thus, topically applied cathepsin B inhibitors might effectively limit DTHR such as contact dermatitis or psoriasis. However, the cathepsin B and Z knockout mouse experiments suggested a complementary role for these two cysteine-type proteases.

Hypersensitivity diseases are associated with many severe human illnesses, including leprosy and tuberculosis. Emerging evidence suggests that the pathogenesis and pathological mechanisms of treating these diseases may be attributable to sphingolipid metabolism.

Mounting evidence suggests mesenchymal stromal cells (MSCs) suppress CD4+ T-cell activation, but whether MSCs directly regulate activation and expansion of allogeneic T cells has not been fully deciphered. Here, we identified that both human and murine MSCs constitutively express ALCAM, a cognate ligand for CD6 receptors on T cells, and investigated its immunomodulatory function using in vivo and in vitro experiments. Our controlled coculture assays demonstrated that ALCAM-CD6 pathway is critical for MSCs to exert its suppressive function on early CD4+CD25- T-cell activation. Moreover, neutralizing ALCAM or CD6 results in the abrogation of MSC-mediated suppression of T-cell expansion. Using a murine model of delayed-type hypersensitivity response to alloantigen, we show that ALCAM-silenced MSCs lose the capacity to suppress the generation of alloreactive IFNγ-secreting T cells. Consequently, MSCs, following ALCAM knockdown, failed to prevent allosensitization and alloreactive T-cell-mediated tissue damage.

The heparan sulfate mimetic PG545 (pixatimod) is under evaluation as an inhibitor of angiogenesis and metastasis including in human clinical trials. We have examined the effects of PG545 on lymphocyte phenotypes and function. We report that PG545 treatment suppresses effector T cell activation and polarizes T cells away from Th17 and Th1 and toward Foxp3+ regulatory T cell subsets in vitro and in vivo. Mechanistically, PG545 inhibits Erk1/2 signaling, a pathway known to affect both T cell activation and subset polarization. Interestingly, these effects are also observed in heparanase-deficient T cells, indicating that PG545 has effects that are independent of its role in heparanase inhibition. Consistent with these findings, administration of PG545 in a Th1/Th17-dependent mouse model of a delayed-type hypersensitivity led to reduced footpad inflammation, reduced Th17 memory cells, and an increase in FoxP3+ Treg proliferation. PG545 also promoted Foxp3+ Treg induction by human T cells. Finally, we examined the effects of other heparan sulfate mimetics PI-88 and PG562 on lymphocyte polarization and found that these likewise induced Foxp3+ Treg in vitro but did not reduce Th17 numbers or improve delayed-type hypersensitivity in this model. Together, these data indicate that PG545 is a potent inhibitor of Th1/Th17 effector functions and inducer of FoxP3+ Treg. These findings may inform the adaptation of PG545 for clinical applications including in inflammatory pathologies associated with type IV hypersensitivity responses.

Pertussis toxin (PTX), an exotoxin of Bordetella pertussis, enhances the development of experimental autoimmune diseases such as experimental autoimmune uveitis (EAU) and experimental autoimmune encephalomyelitis (EAE) in rodent models. The mechanisms of the promotion of experimental autoimmune diseases by PTX may be based upon PTX-induced disruption of the blood eye/brain barriers facilitating the infiltration of inflammatory cells, the modulation of inflammatory cell migration and the enhancement of the activation of inflammatory cells. We hypothesized that the facilitation of experimental autoimmunity by PTX suggests that its influence on the in vivo immune response to auto-antigen may differ from its influence on non-self antigens.