Multiomic single-cell sequencing defines tissue-specific responses in Stevens-Johnson Syndrome and Toxic epidermal necrolysis.

Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) is a rare but life-threatening cutaneous drug reaction mediated by human leukocyte antigen (HLA) class I-restricted CD8+ T-cells. To obtain an unbiased assessment of SJS/TEN cellular immunopathogenesis, we performed single-cell (sc) transcriptome, surface proteome, and TCR sequencing on unaffected skin, affected skin, and blister fluid from 17 SJS/TEN patients. From 119,784 total cells, we identified 16 scRNA-defined subsets, confirmed by subset-defining surface protein expression. Keratinocytes upregulated HLA and IFN-response genes in the affected skin. Cytotoxic CD8+ T-cell subpopulations of expanded and unexpanded TCRαß clonotypes were shared in affected skin and blister fluid but absent or unexpanded in SJS/TEN unaffected skin. SJS/TEN blister fluid is a rich reservoir of oligoclonal CD8+ T-cells with an effector phenotype driving SJS/TEN pathogenesis. This multiomic database will act as the basis to define antigen-reactivity, HLA restriction, and signatures of drug-antigen-reactive T-cell clonotypes at a tissue level.

Skin infiltrating T-cell profile of drug reaction with eosinophilia and systemic symptoms (DRESS) reactions among HIV-infected patients.

Introduction: Drug Reaction with Eosinophilia Systemic Symptoms (DRESS) is more common in persons living with HIV (PLHIV), and first-line anti-TB drugs (FLTDs) and cotrimoxazole are the commonest offending drugs. Limited data is available on the skin infiltrating T-cell profile among DRESS patients with systemic CD4 T-cell depletion associated with HIV. Materials and methods: HIV cases with validated DRESS phenotypes (possible, probable, or definite) and confirmed reactions to either one or multiple FLTDs and/or cotrimoxazole were chosen (n = 14). These cases were matched against controls of HIV-negative patients who developed DRESS (n = 5). Immunohistochemistry assays were carried out with the following antibodies: CD3, CD4, CD8, CD45RO and FoxP3. Positive cells were normalized to the number of CD3+ cells present. Results: Skin infiltrating T-cells were mainly found in the dermis. Dermal and epidermal CD4+ T-cells (and CD4+/CD8+ ratios) were lower in HIV-positive vs. negative DRESS; p < 0.001 and p = 0.004, respectively; without correlation to whole blood CD4 cell counts. In contrast, no difference in dermal CD4+FoxP3+ T-cells was found in HIV-positive vs. negative DRESS, median (IQR) CD4+FoxP3+ T-cells: [10 (0-30) cells/mm2 vs. 4 (3-8) cells/mm2, p = 0.325]. HIV-positive DRESS patients reacting to more than one drug had no difference in CD8+ T-cell infiltrates, but higher epidermal and dermal CD4+FoxP3+ T-cell infiltrates compared to single drug reactors. Conclusion: DRESS, irrespective of HIV status, was associated with an increased skin infiltration of CD8+ T-cells, while CD4+ T-cells were lower in HIV-positive DRESS compared to HIV-negative DRESS skin. While inter-individual variation was high, the frequency of dermal CD4+FoxP3+ T-cells was higher in HIV-positive DRESS cases reacting to more than one drug. Further research is warranted to understand the clinical impact of these changes.

HIV-Associated Immune Dysregulation in the Skin: A Crucible for Exaggerated Inflammation and Hypersensitivity.

Skin diseases are hallmarks of progressive HIV-related immunosuppression, with severe noninfectious inflammatory and hypersensitivity conditions as common as opportunistic infections. Conditions such as papular pruritic eruption are AIDS defining, whereas delayed immune-mediated adverse reactions, mostly cutaneous, occur up to 100-fold more during HIV infection. The skin, constantly in contact with the external environment, has a complex immunity. A dense, tightly junctioned barrier with basal keratinocytes and epidermal Langerhans cells with antimicrobial, innate-activating, and antigen-presenting functions form the frontline. Resident dermal dendritic, mast, macrophage, and innate lymphoid cells play pivotal roles in directing and polarizing appropriate adaptive immune responses and directing effector immune cell trafficking. Sustained viral replication leads to progressive declines in CD4 T cells, whereas Langerhans and dermal dendritic cells serve as viral reservoirs and points of first viral contact in the mucosa. Cutaneous cytokine responses and diminished lymphoid populations create a crucible for exaggerated inflammation and hypersensitivity. However, beyond histopathological description, these manifestations are poorly characterized. This review details normal skin immunology, changes associated with progressive HIV-related immunosuppression, and the characteristic conditions of immune dysregulation increased with HIV. We highlight the main research gaps and several novel tissue-directed strategies to define mechanisms that will provide targeted approaches to prevention or treatment.

IFN-γ ELISpot in Severe Cutaneous Adverse Reactions to First-Line Antituberculosis Drugs in an HIV Endemic Setting.

Severe cutaneous adverse reactions related to first-line antituberculosis drugs are associated with high mortality and long-term morbidity. Oral sequential drug challenge, as a form of drug provocation testing, helps to salvage therapy by identifying culprit drugs but is associated with risk and is costly. IFN-γ enzyme-linked immune absorbent spot (ELISpot), an adjunctive in vitro diagnostic tool, may help to guide risk-stratification approaches. To determine the diagnostic accuracy of IFN-γ ELISpot against full-dose sequential drug challenge, we analyzed samples collected prospectively at multiple time points in 32 patients with first-line antituberculosis drug‒associated severe cutaneous adverse reaction (81% HIV infected, 25 with drug reaction with eosinophilia and systemic symptoms, and 7 with Stevens‒Johnson syndrome/toxic epidermal necrolysis). Sensitivity of IFN-γ ELISpot was 33% (4 of 12), 13% (1 of 8), 11% (1 of 9), and 0% (0 of 4) for rifampicin, isoniazid, pyrazinamide, and ethambutol, respectively (positivity threshold ≥50 spot forming units/million cells). Specificity was 100% for all the four drugs. Rifampicin IFN-γ ELISpot sensitivity increased to 58% (7 of 12) if a threshold of 20 spot forming units was used and to 75% (3 of 4) when restricted to samples <12 weeks after acute severe cutaneous adverse reaction event; specificity remained 100% for both. IFN-γ ELISpot offers adequate risk stratification of rifampicin severe cutaneous adverse reaction using acute samples and lowered threshold for positivity. Given the low sensitivity of IFN-γ ELISpot for other first-line antituberculosis drugs, additional optimization is needed to improve risk-stratification potential.

Corrigendum: Dose Dependent Antimicrobial Cellular Cytotoxicity-Implications for ex vivo Diagnostics.

[This corrects the article DOI: 10.3389/fphar.2021.640012.].

Dose Dependent Antimicrobial Cellular Cytotoxicity-Implications for ex vivo Diagnostics.

Introduction: Ex vivo and in vitro diagnostics, such as interferon-γ (IFN-γ) release enzyme linked ImmunoSpot (ELISpot) and flow cytometry, are increasingly employed in the research and diagnostic setting for severe T-cell mediated hypersensitivity. Despite an increasing use of IFN-γ release ELISpot for drug causality assessment and utilization of a range of antimicrobial concentrations ex vivo, data regarding antimicrobial-associated cellular cytotoxicity and implications for assay performance remain scarcely described in the literature. Using the measurement of lactate dehydrogenase (LDH) and the 7-AAD cell viability staining, we aimed via an exploratory study, to determine the maximal antimicrobial concentrations required to preserve cell viability for commonly implicated antimicrobials in severe T-cell mediated hypersensitivity. Method: After an 18-h incubation of patient peripheral blood monocytes (PBMCs) and antimicrobials at varying drug concentrations, the cell cytotoxicity was measured in two ways. A colorimetric based assay that detects LDH activity and by flow cytometry using the 7-AAD cell viability staining. We used the PBMCs collected from three healthy control participants with no known history of adverse drug reaction and two patients with a rifampicin-associated drug reaction with eosinophilia and systemic symptoms (DRESS), confirmed on IFN-γ ELISpot assay. The PBMCs were stimulated for the investigated drugs at the previously published drug maximum concentration (Cmax), and concentrations 10- and 100-fold above. Results: In a human immunodeficiency virus (HIV) negative and a positive rifampicin-associated DRESS with positive ex vivo IFN-γ ELISpot assay, use of 10- and 100-fold Cmax drug concentrations decreased spot forming units/million cells by 32-100%, and this corresponded to cell cytotoxicity of more than 40 and 20% using an LDH assay and 7-AAD cell viability staining, respectively. The other antimicrobials (ceftriaxone, flucloxacillin, piperacillin/tazobactam, and isoniazid) tested in healthy controls showed similar dose-dependent increased cytotoxicity using the LDH assay, but cytotoxicity remained lower than 40% for all Cmax and 10-fold Cmax drug concentrations except flucloxacillin. All 100-fold Cmax concentrations resulted in cell death >40% (median 57%), except for isoniazid. 7-AAD cell viability staining also confirmed an increase in lymphocyte death in PBMCs incubated with 10-fold and 100-fold above Cmax for ceftriaxone, and flucloxacillin; however, piperacillin/tazobactam and isoniazid indicated no differences in percentages of viable lymphocytes across concentrations tested. Conclusion: The LDH cytotoxicity and 7-AAD cell viability staining techniques both demonstrate increased cell death corresponding to a loss in ELISpot sensitivity, with use of higher antimicrobial drug concentrations for ex vivo diagnostic IFN-γ ELISpot assays. For all the antimicrobials evaluated, the use of Cmax and 10-fold Cmax concentrations impacts cell viability and potentially affects ELISpot performance. These findings inform future approaches for ex vivo diagnostics such as IFN-γ release ELISpot.

Drug hypersensitivity in HIV infection.

PURPOSE OF REVIEW: Immune-mediated adverse drug reactions (IM-ADRs) are many times more common in HIV-infected patients. Usual offending drugs include antiretroviral and antiinfectives, but the burden of specific drug IM-ADRs is population-specific; changing as new and fixed dose combinations enter the market, and drug-resistance patterns demand. This review considers recent literature on epidemiology, mechanisms, clinical management and prevention of IM-ADRs amongst persons living with HIV/AIDS. RECENT FINDINGS: Epidemiological studies continue to describe high rates of delayed hypersensitivity to known offenders, as well as similar reactions in preexposure prophylaxis. IM-ADRs to oral and injectable integrase strand transfer inhibitors are reported with expanding use. The clinical spectrum and management of IM-ADRs occurring in HIV-infected populations is similar to uninfected; with exceptions such as a recently described severe delayed efavirenz DILI with high mortality. Furthermore, the context can be unique, such as the lower than expected mortality in a Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) cohort from a HIV/TB high burden setting. Programmatic data showing the near complete elimination of Abacavir drug hypersensitivity syndrome following implementation of HLA-B57:01 screening is a stellar example of how prevention is possible with mechanistic insight. SUMMARY: IM-ADRs remain a challenge in persons living with HIV. The complexities posed by polypharmacy, overlapping drug toxicities, drug interactions, overlap of IM-ADRs with other diseases, limited alternative drugs, and vulnerable patients with advanced immunosuppression with high mortality, necessitate increased use of drug provocation testing, treat-through and desensitization strategies. There is an urgent need for improved diagnostics and predictive biomarkers for prevention, or to guide treat-through, rechallenge and desensitization approaches.