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The peptidic nature of the male (H-Y) antigen, a model minor histocompatibility antigen in H-2b mice, has recently been demonstrated. In this study we show that the H-Y peptide, which is recognized by PM-1, a Db-restricted cytotoxic T-lymphocyte (CTL) clone, is absent in male H-2d spleen cells but present in male H-2d spleen cells that also express a transgenic Db molecule under its endogenous promoter. This result indicates that both the H-Y and the Db gene products are essential and sufficient for production of the Db-restricted H-Y peptide. By comparing the ability of the PM-1 clone and bulk CTL generated in a secondary mixed lymphocyte culture to recognize H-Y peptidic material eluted from affinity-purified Db molecules and separated by reversed-phase high-performance liquid chromatography (HPLC), we provide evidence that there is an immunodominant H-Y epitope that is presented by the Db molecule. Furthermore, the presentation of this epitope is not affected by a mutation in the α3 domain of Db (asp227 to lys227), which abrogates CD8 binding, since similar amounts of H-Y peptide were eluted from affinity-purified wild-type or mutant Db molecules. However, the generation of the H-Y epitope is dependent on the presence of β2-microglobulin, since it is absent in male H-2b mice that lack a functional β2microglobulin gene. The implications of these findings on T-cell development are discussed. [ABSTRACT FROM AUTHOR]

Abstract Background In addition to progressive CD4+ T cell immune deficiency, HIV infection is characterized by generalized immune activation, thought to arise from increased microbial exposure resulting from diminishing immunity. Results Here we report that, in a virus-free mouse model, conditional ablation of activated CD4+ T cells, the targets of immunodeficiency viruses, accelerates their turnover and produces CD4+ T cell immune deficiency. More importantly, activated CD4+ T cell killing also results in generalized immune activation, which is attributable to regulatory CD4+ T cell insufficiency and preventable by regulatory CD4+ T cell reconstitution. Immune activation in this model develops independently of microbial exposure. Furthermore, microbial translocation in mice with conditional disruption of intestinal epithelial integrity affects myeloid but not T cell homeostasis. Conclusions Although neither ablation of activated CD4+ T cells nor disruption of intestinal epithelial integrity in mice fully reproduces every aspect of HIV-associated immune dysfunction in humans, ablation of activated CD4+ T cells, but not disruption of intestinal epithelial integrity, approximates the two key immune alterations in HIV infection: CD4+ T cell immune deficiency and generalized immune activation. We therefore propose activated CD4+ T cell killing as a common etiology for both immune deficiency and activation in HIV infection. See minireview http://www.jbiol.com/content/8/10/91

Drug-checking services (DCS) provide people who use drugs (PWUD) the opportunity to have their substances tested before consumption. Though some suggest they may have adverse consequences, DCS have been introduced as a harm reduction (HR) strategy. A systematized review of the literature regarding drug checking (DC) methods and testing locations, advantages and disadvantages, and legal frameworks with an emphasis on HR was conducted referencing PRISMA guidelines. The primary search of PsychInfo, PubMed, Medline, CINHAL, CORE, and Web of Science was conducted between the 4 th and 10 th of September 2020, and 51 literature pieces were included in the final article. Most of the literature focuses on the benefits of currently available DCS. The services identified varied significantly in terms of testing methods, location of operation, primary goal, and the surrounding legal framework. The results suggest using multiple DC methods to be most beneficial. Further, DCS and the personalized interventions they provide can positively influence behavior change, minimize harm, and reduce mortality. DCS are a viable public health intervention that requires cross-sector support beyond the legal frameworks and testing methods. Services will need to be tailored to meet the needs of their chosen setting, local drug market, and target audience.

Background: Festival drug-related deaths are a growing public health concern., Aim: To examine drug use and related harm-reduction practices and attitudes towards utilisation of drug safety testing services., Methods: Data collection took place over the 2019 festival season (June-October). The questionnaire was self-reported. Data was gathered via the online survey, which was promoted through online and social media platforms and outlets. Social media communication methods were used to reach the targeted population more effectively., Results: A total of 1193 Irish festival attendees over the age of 18 completed an anonymous online survey. Alcohol, MDMA powder/crystals, ecstasy pills and cocaine were the highest reported drugs used by Irish festival attendees. The vast majority of participants reported polysubstance use (86.8%/n = 1036). Forty percent of participants (39.98%/n = 477) reported having had sex following the use of a drug at a festival; of these, 66% (n = 316) said that the sex was unprotected. Most participants (84.0%/n = 1003) engaged in some form of harm reduction when taking drugs at festivals. Overwhelmingly, participants reported a willingness to engage with drug-checking services. The vast majority (96.3%; n = 1149) and would use drug checking services more than three-quarters (75.1%/n = 897) reported that they would use an 'amnesty bin' for drugs if it were part of an alert system to notify if dangerous drugs are in circulation. A chi-square test of Independence was conducted to examine whether age and utilisation of drug safety testing service a festival were independent. Moreover, when all cases are taken together, the difference between testing modalities (onsite, offsite and amnesty bin) shows a significant difference p < 001 between those who would use onsite and offsite drug testing facilities., Conclusion: The evidence from this survey indicates that those young people who use drugs at festivals would be prepared to utilise drug checking services and amnesty bins should help inform the public health response to this important area., (© 2021. The Author(s).)

Background: Emergency contingency guidelines for opioid agonist treatment (OAT) were introduced in Ireland in March 2020, to ensure rapid and uninterrupted access to treatment while mitigating COVID-19 risk. The contingency guidelines deviated, across multiple clinical domains, from pre-pandemic clinical guidelines published in 2016. The objectives of this study are to (1) identify changes introduced to OAT clinical guidelines in Ireland during the pandemic; and (2) develop consensus on whether the new recommendations should be retained beyond the pandemic, using a national Delphi consensus methodology., Methods: Clinical guidance recommendations ('statements') were generated by comparing the newly established contingency guidelines with the national 2016 Clinical Guidelines for OAT. Over two rounds of on-line Delphi testing, a panel of experts (people currently accessing OAT, psychiatrists, general practitioners, community pharmacists, a nurse, a psychologist and support/key workers) independently rated their agreement with each statement and provided comments. Statements with a median score of 4 or 5 and a lower quartile of ≥4 were classified as having reached consensus., Results: Forty-eight panel members were recruited, with a high participation level at Round 2 (90%, n=43). Consensus was achieved for 12 of the 19 statements at Round 1. The 7 remaining statements were revised, with 2 new statements, resulting in 9 statements at Round 2. Four statements reached consensus at Round 2. The final list includes 16 clinical guidance statements; 9 relating to assessment, 3 to OAT drug choice and dosing, 1 to take-away doses, 2 to overdose prevention and 1 to the continuation of e-prescriptions., Conclusions: A wide range of stakeholders involved in the delivery and receipt of OAT agreed on 16 clinical guidance statements for inclusion in OAT clinical guidelines as we move beyond the pandemic, rather than reverting to pre-pandemic guidelines. The agreed statements relate to facilitating safe access to OAT with minimal waiting time, supporting patient-centred care to promote health and well-being, and preventing drug overdose. Notably, consensus was not achieved for OAT drug dosage and frequency of urine testing during the stabilisation and maintenance phase of care., Competing Interests: Declarations of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Current approaches to reducing the latent HIV reservoir entail first reactivating virus-containing cells to become visible to the immune system. A critical second step is killing these cells to reduce reservoir size. Endogenous cytotoxic T-lymphocytes (CTLs) may not be adequate because of cellular exhaustion and the evolution of CTL-resistant viruses. We have designed a universal CAR-T cell platform based on CTLs engineered to bind a variety of broadly neutralizing anti-HIV antibodies. We show that this platform, convertibleCAR-T cells, effectively kills HIV-infected, but not uninfected, CD4 T cells from blood, tonsil, or spleen and only when armed with anti-HIV antibodies. convertibleCAR-T cells also kill within 48 h more than half of the inducible reservoir found in blood of HIV-infected individuals on antiretroviral therapy. The modularity of convertibleCAR-T cell system, which allows multiplexing with several anti-HIV antibodies yielding greater breadth and control, makes it a promising tool for attacking the latent HIV reservoir., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Competing Interests: Competing interests: None declared.

The generation of allergen-specific TCR transgenic animals allows for the characterization of allergen-specific T-cell responses in vivo and in vitro and is a powerful tool to study adaptive immunity to allergens. Here we describe an approach starting from the isolation of antigen-specific T-cell hybridomas and using PCR, flow cytometric, and co-culture methods to obtain antigen-specific MHC class II-restricted CD4 + TCR transgenic mice on the Rag2 -/- background.

Introduction: A multicentre study (including four cities in Pakistan) aimed to investigate the frequency and spectrum of alpha and beta thalassemia genetic mutations and XmnI polymorphism of the Gamma Globin gene., Methods: One hundred and sixty one beta thalassemia patients, identified on the ground of haematological parameters, were screened for mutations of the Alpha (HBA2 and HBA1) and Beta (HBB) Globin genes as well as Gamma (HBG2) Globin gene, -158 Gγ XmnI polymorphism, using a combination of multiplex GAP polymerase chain reaction (PCR), Sanger sequencing and restriction fragment length polymerase (RFLP) based PCR., Results: Mutations of at least one HBB gene was identified in 157 of 161 patients screened. Among 16 identified mutations in the beta gene, HBB:c.27&#95;28insG (p. Ser10Valfs*14) was the most prevalent. α(-3.7) and α(-4.2) deletions were co-inherited with beta thalassemia mutations. Rare mutations such as HBB:c.-138C > T and HBB:c.315 + 1G > A were also identified. One novel variant (HBB:c.-148T > A), two rare mutations [HBB:c.332T > C (p.Leu111Pro); HBB:c.92G > C (p.Arg31Thr] and a novel association, HBB:c.[92G > C (p.Arg31Thr)] and [-92C > G], were reported for the first time in our study. HBG2:c.-211C > T base-pair substitution (historically described as -158 GγXmnI polymorphism) was present in 36% of the patients., Conclusion: Heterogeneity in clinical and haematological parameters in TM, show that monogenic disorders can present with a wide spectrum of disease severity. Our studies identified rare and novel mutations that will be useful in the prevention of highly prevalent disease of thalassemia in Pakistan following nationwide awareness campaign., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Dendritic cells (DCs) are crucial for mounting allergic airway inflammation, but it is unclear which subset of DCs performs this task. By using CD64 and MAR-1 staining, we reliably separated CD11b(+) monocyte-derived DCs (moDCs) from conventional DCs (cDCs) and studied antigen uptake, migration, and presentation assays of lung and lymph node (LN) DCs in response to inhaled house dust mite (HDM). Mainly CD11b(+) cDCs but not CD103(+) cDCs induced T helper 2 (Th2) cell immunity in HDM-specific T cells in vitro and asthma in vivo. Studies in Flt3l(-/-) mice, lacking all cDCs, revealed that moDCs were also sufficient to induce Th2 cell-mediated immunity but only when high-dose HDM was given. The main function of moDCs was the production of proinflammatory chemokines and allergen presentation in the lung during challenge. Thus, we have identified migratory CD11b(+) cDCs as the principal subset inducing Th2 cell-mediated immunity in the LN, whereas moDCs orchestrate allergic inflammation in the lung., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The human CD2 (hCD2) locus control region (LCR) inserted in the mouse CD8 gene complex activates expression of the CD8 genes in T cell subsets in which the CD8 locus is normally silenced (e.g., CD4(+) single-positive T cells). In this article, we show that, in conditional mCD8/hCD2-LCR (CD8/LCR) knock-in mice, the continuous presence of the hCD2-LCR is required for this effect. Deletion of the inserted hCD2-LCR in a developmental stage and cell lineage-specific manner revealed that the temporary presence of the LCR during early development does not permanently alter the expression pattern of the CD8 genes. As a result, cells that have been affected by the insertion of the LCR can convert to their destined phenotype once the LCR is removed. DNaseI hypersensitive sites 1 and 2 of the hCD2-LCR influence the expression of the CD8 genes in a similar manner as does the full LCR, whereas insertion of hypersensitive site 3 alone of the LCR does not result in a changed expression pattern. This analysis revealed a dynamic interaction between the hCD2-LCR and the endogenous regulatory elements of the CD8 genes.

To investigate the role of the kinase Zap70 in T cells, we generated mice expressing a Zap70 mutant whose catalytic activity can be selectively blocked by a small-molecule inhibitor. We found that conventional naive, effector and memory T cells were dependent on the kinase activity of Zap70 for their activation, which demonstrated a nonredundant role for Zap70 in signals induced by the T cell antigen receptor (TCR). In contrast, the catalytic activity of Zap70 was not required for activation of the GTPase Rap1 and inside-out signals that promote integrin adhesion. This Zap70 kinase-independent pathway was sufficient for the suppressive activity of regulatory T cells (T(reg) cells), which was unperturbed by inhibition of the catalytic activity of Zap70. Our results indicate Zap70 is a likely therapeutic target.

The generation of high-affinity Abs is essential for immunity and requires collaboration between B and T cells within germinal centers (GCs). By using novel mouse models with a conditional deletion of the p110δ catalytic subunit of the PI3K pathway, we established that p110δ is required in T cells, but not in B cells, for the GC reaction. We found the formation of T follicular helper (T(FH)) cells to be critically dependent on p110δ in T cells. Furthermore, by deleting phosphatase and tensin homolog deleted on chromosome 10, which opposes p110δ in activated T cells, we found a positive correlation between increased numbers of T(FH) cells and GC B cells. These results are consistent with the hypothesis that T cell help is the limiting factor in the GC reaction. P110δ was not required for the expression of B cell lymphoma 6, the downregulation of CCR7, or T cell entry into primary follicles. Instead, p110δ was the critical catalytic subunit for ICOS downstream signaling and the production of key T(FH) cytokines and effector molecules. Our findings support a model in which the magnitude of the GC reaction is controlled by the activity of the PI3K pathway in T(FH) cells.

Many functions of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) have been defined, but relatively little is known about the biology of an alternative mTOR complex, mTORC2. We showed that conditional deletion of rictor, an essential subunit of mTORC2, impaired differentiation into T helper 1 (Th1) and Th2 cells without diversion into FoxP3(+) status or substantial effect on Th17 cell differentiation. mTORC2 promoted phosphorylation of protein kinase B (PKB, or Akt) and PKC, Akt activity, and nuclear NF-kappaB transcription factors in response to T cell activation. Complementation with active Akt restored only T-bet transcription factor expression and Th1 cell differentiation, whereas activated PKC-theta reverted only GATA3 transcription factor and the Th2 cell defect of mTORC2 mutant cells. Collectively, the data uncover vital mTOR-PKC and mTOR-Akt connections in T cell differentiation and reveal distinct pathways by which mTORC2 regulates development of Th1 and Th2 cell subsets., (Copyright 2010 Elsevier Inc. All rights reserved.)

Monosomy 7 and del(7q) are associated with adverse features in myeloid malignancies. A 2.5-Mb commonly deleted segment (CDS) of chromosome band 7q22 is implicated as harboring a myeloid tumor suppressor gene (TSG); however, molecular analysis of candidate TSGs has not uncovered loss of function. To determine whether haploinsufficiency for the 7q22 CDS contributes to myeloid leukemogenesis, we performed sequential gene targeting to flank a region of orthologous synteny on mouse chromosome band 5A3 with loxP sites. We then generated Mx1-Cre, 5A3(fl) mutant mice and deleted the targeted interval in vivo. Although excision was inefficient, we confirmed somatic deletion of the 5A3 CDS in the hematopoietic stem cell compartment. Mx1-Cre, 5A3(fl) mice show normal hematologic parameters and do not spontaneously develop myeloid malignancies. The 5A3(fl) deletion does not cooperate with oncogenic Kras(G12D) expression, Nf1 inactivation, or retroviral mutagenesis to accelerate leukemia development and did not modulate responsiveness to antileukemia drugs. These studies demonstrate that it is feasible to somatically delete a large chromosomal segment implicated in tumor suppression in hematopoietic cell populations in vivo; however, our data do not support the hypothesis that the 7q22/5A3 CDS interval contains a myeloid TSG.

Alleles that express reporters after Cre recombination allow for fate-mapping studies when used in combination with appropriate cre alleles. In this study, we describe two fluorescent reporter alleles that differentially mark populations of cells as a function of their level of expression of Cre recombinase. Mice carrying these alleles were generated and used to demonstrate the usefulness of the reporter alleles for informing on prior Cre recombinase expression in lymphocytes. The alleles expand the range of genetic tools available for understanding how differences in gene expression result in divergent developmental fates during the development and differentiation of lymphocytes and other cells.

Signaling through the T cell antigen receptor (TCR) is important for the homeostasis of naïve and memory CD4(+) T cells. The significance of TCR signaling in regulatory T (Treg) cells has not been systematically addressed. Using an Ox40-cre allele that is prominently expressed in Treg cells, and a conditional null allele of the gene encoding p56(Lck), we have examined the importance of TCR signaling in Treg cells. Inactivation of p56(Lck) resulted in abnormal Treg homeostasis characterized by impaired turnover, preferential redistribution to the lymph nodes, loss of suppressive function, and striking changes in gene expression. Abnormal Treg cell homeostasis and function did not reflect the involvement of p56(Lck) in CD4 function because these effects were not observed when CD4 expression was inactivated by Ox40-cre.The results make clear multiple aspects of Treg cell homeostasis and phenotype that are dependent on a sustained capacity to signal through the TCR.

Protein disulfide isomerases (PDIs) aid protein folding and assembly by catalyzing formation and shuffling of cysteine disulfide bonds in the endoplasmic reticulum (ER). Many members of the PDI family are expressed in mammals, but the roles of specific PDIs in vivo are poorly understood. A recent homology-based search for additional PDI family members identified anterior gradient homolog 2 (AGR2), a protein originally presumed to be secreted by intestinal epithelial cells. Here, we show that AGR2 is present within the ER of intestinal secretory epithelial cells and is essential for in vivo production of the intestinal mucin MUC2, a large, cysteine-rich glycoprotein that forms the protective mucus gel lining the intestine. A cysteine residue within the AGR2 thioredoxin-like domain forms mixed disulfide bonds with MUC2, indicating a direct role for AGR2 in mucin processing. Mice lacking AGR2 were viable but were highly susceptible to colitis, indicating a critical role for AGR2 in protection from disease. We conclude that AGR2 is a unique member of the PDI family, with a specialized and nonredundant role in intestinal mucus production.

OX40 is a member of the TNF receptor family expressed on activated and regulatory T (Treg) cells. Using an Ox40-cre allele for lineage marking, we found that a subpopulation of naive T cells had also previously expressed OX40 in the thymus. Ox40-cre was induced in a small fraction of thymocytes that were OX40(+), some of which were CD25(high) Treg cell precursors. Thymic OX40 expression distinguished cells experiencing a strong signaling response to positive selection. Naive T cells that had previously expressed OX40 demonstrated a partially activated phenotype that was distinct from that of most naive T cells. The results are consistent with the selection of Treg cells and a minor subpopulation of naive T cells being dependent on strong signaling responses to thymic self ligands.