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Neuromuscular blocking agents (NMBAs) relax skeletal muscles to facilitate surgeries and ease intubation but can lead to adverse reactions, including complications because of postoperative residual neuromuscular blockade (rNMB) and, in rare cases, anaphylaxis. Both adverse reactions vary between types of NMBAs, with rocuronium, a widely used nondepolarizing NMBA, inducing one of the longest rNMB durations and highest anaphylaxis incidences. rNMB induced by rocuronium can be reversed by the synthetic γ-cyclodextrin sugammadex. However, in rare cases, sugammadex can provoke anaphylaxis. Thus, additional therapeutic options are needed. Rocuronium-induced anaphylaxis is proposed to rely on preexisting rocuronium-binding antibodies. To understand the pathogenesis of rocuronium-induced anaphylaxis and to identify potential therapeutics, we investigated the memory B cell antibody repertoire of patients with suspected hypersensitivity to rocuronium. We identified polyclonal antibody repertoires with a high diversity among V(D)J genes without evidence of clonal groups. When recombinantly expressed, these antibodies demonstrated specificity and low affinity for rocuronium without cross-reactivity for other NMBAs. Moreover, when these antibodies were expressed as human immunoglobulin E (IgE), they triggered human mast cell activation and passive systemic anaphylaxis in transgenic mice, although their affinities were insufficient to serve as reversal agents. Rocuronium-specific, high-affinity antibodies were thus isolated from rocuronium-immunized mice. The highest-affinity antibody was able to reverse rocuronium-induced neuromuscular blockade in nonhuman primates with kinetics comparable to that of sugammadex. Together, these data support the hypothesis that antibodies cause anaphylactic reactions to rocuronium and pave the way for improved diagnostics and neuromuscular blockade reversal agents. Editor's summary: Neuromuscular blocking agents (NMBAs) such as rocuronium are used during anesthesia to assist with surgeries and intubations. Although these agents are effective, they are not without risk. Patients can develop postoperative residual neuromuscular blockade, which can be treated with an NMBA reversal agent, and life-threatening anaphylaxis can occur in rare cases. Here, Dejoux et al. isolated and characterized memory B cells from three individuals who developed reactions to NMBA treatment and had serum antibodies to rocuronium. The authors found that IgE antibodies generated from these memory B cells were sufficient to induce anaphylaxis in mice treated with rocuronium, although they were too low affinity to be used as reversal agents. This led the authors to immunize mice with rocuronium to generate antibodies of higher affinity, which were able to reverse neuromuscular blockade in rocuronium-treated nonhuman primates. Together, these data explain why these three patients had a reaction and offer a therapeutic approach to reversing neuromuscular blockade. —Courtney Malo [ABSTRACT FROM AUTHOR]

Flow cytometry is the method of choice for immunophenotyping in the context of clinical, translational, and systems immunology studies. Among the latter, the Milieu Intérieur (MI) project aims at defining the boundaries of a healthy immune response to identify determinants of immune response variation. MI used immunophenotyping of a 1000 healthy donor cohort by flow cytometry as a principal outcome for immune variance at steady state. New generation spectral cytometers now enable high‐dimensional immune cell characterization from small sample volumes. Therefore, for the MI 10‐year follow up study, we have developed two high‐dimensional spectral flow cytometry panels for deep characterization of innate and adaptive whole blood immune cells (35 and 34 fluorescent markers, respectively). We have standardized the protocol for sample handling, staining, acquisition, and data analysis. This approach enables the reproducible quantification of over 182 immune cell phenotypes at a single site. We have applied the protocol to discern minor differences between healthy and patient samples and validated its value for application in immunomonitoring studies. Our protocol is currently used for characterization of the impact of age and environmental factors on peripheral blood immune phenotypes of >400 donors from the initial MI cohort. [ABSTRACT FROM AUTHOR]

Stunting and environmental enteric dysfunction (EED) may be responsible for altered gut and systemic immune responses. However, their impact on circulating immune cell populations remains poorly characterized during early life. A detailed flow cytometry analysis of major systemic immune cell populations in 53 stunted and 52 non-stunted (2 to 5 years old) children living in Antananarivo (Madagascar) was performed. Compared to age-matched non-stunted controls, stunted children aged 2-3 years old had a significantly lower relative proportion of classical monocytes. No significant associations were found between stunting and the percentages of effector T helper cell populations (Th1, Th2, Th17, Th1Th17, and cTfh). However, we found that HLA-DR expression (MFI) on all memory CD4+ or CD8+ T cell subsets was significantly lower in stunted children compared to non-stunted controls. Interestingly, in stunted children compared to the same age-matched non-stunted controls, we observed statistically significant agespecific differences in regulatory T cells (Treg) subsets. Indeed, in 2- to 3-year-old stunted children, a significantly higher percentage of memory Treg, whilst a significantly lower percentage of naive Treg, was found. Our results revealed that both innate and adaptive systemic cell percentages, as well as activation status, were impacted in an agerelated manner during stunting. Our study provides valuable insights into the understanding of systemic immune system changes in stunted children. [ABSTRACT FROM AUTHOR]

HFE, an MHC class Ib molecule that controls iron metabolism, can be directly targeted by cytotoxic TCR αβ T lymphocytes. Transgenic DBA/2 mice expressing, in a Rag 2 KO context, an αβ TCR that directly recognizes mouse HFE (m HFE) were created to further explore the interface of HFE with the immune system. TCR-transgenic m Hfe WT mice deleted m HFE-reactive T cells in the thymus, but a fraction of reprogrammed cells were able to escape deletion. In contrast, TCR-transgenic mice deprived of m HFE molecules (m Hfe KO mice) or expressing a C282→ Y mutated m HFE molecule - the most frequent mutation associated with human hereditary hemochromatosis - positively selected m HFE-reactive CD8+ T lymphocytes and were not tolerant toward m HFE. By engrafting these mice with DBA/2 WT (m HFE+) skin, it was established, as suspected on the basis of similar engraftments performed on DBA/2 m Hfe KO mice, that m HFE behaves as an autonomous skin-associated histocompatibility antigen, even for m HFE- C282→ Y mutated mice. By contrast, infusion of DBA/2 m HFE+ mice with naïve m HFE-reactive transgenic CD8+ T lymphocytes did not induce GVHD. Thus, tolerance toward HFE in m Hfe WT mice can be acquired at either thymic or peripheral levels but is disrupted in mice reproducing human familial hemochromatosis. [ABSTRACT FROM AUTHOR]

In most cells, the NF-κB transcription factor is sequestered in the cytoplasm by interaction with inhibitory proteins, the IκBs. Here, we show that combined IκBα/IκBe deficiency in mice leads to neonatal death, elevated κB binding activity, overexpression of NF-κB target genes, and disruption of lymphocyte production. In IκBα/IκBs-deficient fetuses, B220[SUP+]IgM[SUP+] B cells and single-positive T cells die by apoptosis. In adults, IκBα[SUP-/-]IκBe[SUP-/-] reconstituted chimeras exhibit a nearly complete absence of T and B cells that is not rescued by cotransfer with wild-type bone marrow. These findings demonstrate that IκBs tightly control NF-κB activity in vivo and that increased NF-κB activity intrinsically impairs lymphocyte survival. Because reduction or rise of NF-κB activity leads to similar dysfunction, they also reveal that only a narrow window of NF-κB activity is tolerated by lymphocytes. [ABSTRACT FROM AUTHOR]

dsRNA-dependent protein kinase R (PKR) is a key factor of innate immunity. It is involved in translation inhibition, apoptosis, and enhancement of the proinflammatory and IFN responses. However, how these antiviral functions are conserved during evolution remains largely unknown. Overexpression and knockout studies in a Chinook salmon (Oncorhynchus tshawytscha) cell line were conducted to assess the role of salmonid PKR in the antiviral response. Three distinct mRNA isoforms from a unique pkr gene, named pkr-fl (full length), pkr-ml (medium length) and pkr-sl (short length), were cloned and a pkr-/- clonal fish cell line was developed using CRISPR/Cas9 genome editing. PKR-FL includes an N-terminal dsRNA-binding domain and a C-terminal kinase domain, whereas PKR-ML and PKR-SL display a truncated or absent kinase domain, respectively. PKR-FL is induced during IFNA2 stimulation but not during viral hemorrhagic septicemia virus (VHSV) infection. Overexpression experiments showed that only PKR-FL possesses antiviral functions, including activation of apoptosis and inhibition of de novo protein synthesis. Knockout experiments confirmed that PKR is involved in apoptosis activation during the late stage of VHSV infection. Endogenous PKR also plays a critical role in translation inhibition upon poly(I:C) transfection after IFNA2 treatment. It is, however, not involved in translational arrest during VHSV infection. Extra- and intracellular titrations showed that endogenous PKR does not directly inhibit viral replication but apparently favors virion release into the supernatant, likely by triggering late apoptosis. Altogether, our data confirm that salmonid PKR has conserved molecular functions that VHSV appears to bypass with subversion strategies., (Copyright © 2024 by The American Association of Immunologists, Inc.)

HFE, an MHC class Ib molecule that controls iron metabolism, can be directly targeted by cytotoxic TCR αβ T lymphocytes. Transgenic DBA/2 mice expressing, in a Rag 2 KO context, an αβ TCR that directly recognizes mouse HFE (mHFE) were created to further explore the interface of HFE with the immune system. TCR-transgenic mHfe WT mice deleted mHFE-reactive T cells in the thymus, but a fraction of reprogrammed cells were able to escape deletion. In contrast, TCR-transgenic mice deprived of mHFE molecules (mHfe KO mice) or expressing a C282→Y mutated mHFE molecule - the most frequent mutation associated with human hereditary hemochromatosis - positively selected mHFE-reactive CD8(+) T lymphocytes and were not tolerant toward mHFE. By engrafting these mice with DBA/2 WT (mHFE(+)) skin, it was established, as suspected on the basis of similar engraftments performed on DBA/2 mHfe KO mice, that mHFE behaves as an autonomous skin-associated histocompatibility antigen, even for mHFE-C282→Y mutated mice. By contrast, infusion of DBA/2 mHFE(+) mice with naïve mHFE-reactive transgenic CD8(+) T lymphocytes did not induce GVHD. Thus, tolerance toward HFE in mHfe WT mice can be acquired at either thymic or peripheral levels but is disrupted in mice reproducing human familial hemochromatosis., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)