Superantigens (SAgs) are potent exotoxins secreted by Staphylococcus aureus and Streptococcus pyogenes. They target a large fraction of T cell pools to set in motion a “cytokine storm” with severe and sometimes life-threatening consequences typically encountered in toxic shock syndrome (TSS). Given the rapidity with which TSS develops, designing timely and truly targeted therapies for this syndrome requires identification of key mediators of the cytokine storm’s initial wave. Equally important, early host responses to SAgs can be accompanied or followed by a state of immunosuppression, which in turn jeopardizes the host’s ability to combat and clear infections. Unlike in mouse models, the mechanisms underlying SAg-associated immunosuppression in humans are ill-defined. In this work, we have identified a population of innate-like T cells, called mucosa-associated invariant T (MAIT) cells, as the most powerful source of pro-inflammatory cytokines after exposure to SAgs. We have utilized primary human peripheral blood and hepatic mononuclear cells, mouse MAIT hybridoma lines, HLA-DR4-transgenic mice, MAIThighHLA-DR4+ bone marrow chimeras, and humanized NOD-scid IL-2Rγnull mice to demonstrate for the first time that: i) mouse and human MAIT cells are hyperresponsive to SAgs, typified by staphylococcal enterotoxin B (SEB); ii) the human MAIT cell response to SEB is rapid and far greater in magnitude than that launched by unfractionated conventional T, invariant natural killer T (iNKT) or γδ T cells, and is characterized by production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2, but not IL-17A; iii) high-affinity MHC class II interaction with SAgs, but not MHC-related protein 1 (MR1) participation, is required for MAIT cell activation; iv) MAIT cell responses to SEB can occur in a T cell receptor (TCR) Vβ–specific manner but are largely contributed by IL-12 and IL-18; v) as MAIT cells are primed by SAgs, they also begin to develop a molecular signature consistent with exhaustion and failure to participate in antimicrobial defense. Accordingly, they upregulate lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin-3 (TIM-3), and/or programmed cell death-1 (PD-1), and acquire an anergic phenotype that interferes with their cognate function against Klebsiella pneumoniae and Escherichia coli; vi) MAIT cell hyperactivation and anergy co-utilize a signaling pathway that is governed by p38 and MEK1/2. Collectively, our findings demonstrate a pathogenic, rather than protective, role for MAIT cells during infection. Furthermore, we propose a novel mechanism of SAg-associated immunosuppression in humans. MAIT cells may therefore provide an attractive therapeutic target for the management of both early and late phases of severe SAg-mediated illnesses.

No abstract

ObjectiveDrug-resistant (DR) HIV emerges during combined antiretroviral treatment (cART), creating concern about widespread transmission of DR-HIV as cART is expanded in resource-limited countries. The aim of this study was to determine the predominant HIV-1 subtypes and prevalence of transmitted DR mutations among antiretroviral-naïve patients in Iran.DesignTo monitor transmission of DR HIV, a threshold surveillance based on the world health organization (WHO) guidelines was implemented in Iran.MethodsFor this HIVDR threshold surveillance study, blood samples were collected from 50 antiretroviral-naïve HIV-1-infected patients. Antiretroviral-resistant mutations were determined by sequencing HIV-1 protease, reverse transcriptase and integrase regions. The HIV-1 subtype was determined by sequencing the p17 and C2-V5 regions of the gag and env genes, respectively.ResultsPhylogenetic analyses of the sequenced regions revealed that 45 (95.7%) of 47 samples that were successfully obtained were CRF35_AD. The remaining two cases were subtype B (2.1%) and CRF01_AE (2.1%). Consistent results were obtained also from Env and Gag sequences. Regarding prevalence of transmitted DR viruses, two cases were found to harbor reverse transcriptase-inhibitor-resistant mutations (4.3%). In addition, although not in the WHO list for surveillance of transmitted mutations, 13 minor protease-inhibitor-resistant mutations listed in the International AIDS Society-USA panel of drug resistance mutations were found. No DR mutations were detected in the integrase region.ConclusionsOur study clarified that CRF35_AD is the major subtype among HIV-1-infected patients in Iran. According to the WHO categorization method of HIVDR threshold survey, the prevalence of transmitted drug resistant HIV in Iran was estimated as moderate (5–15%).

The interactions between programmed death-1 (PD-1) and its ligands hamper tumor-specific CD8+ T cell (TCD8) responses, and PD-1-based ‘checkpoint inhibitors’ have shown promise in certain cancers, thus revitalizing interest in immunotherapy. PD-1-targeted therapies reverse TCD8 exhaustion/anergy. However, whether they alter the epitope breadth of TCD8 responses remains unclear. This is an important question because subdominant TCD8 are more likely than immunodominant clones to escape tolerance mechanisms and may contribute to protective anticancer immunity. We have addressed this question in an in vivo model of TCD8 responses to well-defined epitopes of a clinically relevant oncoprotein, large T antigen. We found that unlike other co-inhibitory molecules (CTLA-4, LAG-3, TIM-3), PD-1 was highly expressed by subdominant TCD8, which correlated with their propensity to favorably respond to PD-1/PD-L1-blocking antibodies. PD-1 blockade increased the size of subdominant TCD8 clones at the peak of their primary response, and also sustained their presence giving rise to an enlarged memory pool. The expanded population was fully functional as judged by IFN-γ production and MHC I-restricted cytotoxicity. The selective increase in subdominant TCD8 clonal size was due to their enhanced survival, not proliferation. Further mechanistic studies utilizing peptide-pulsed dendritic cells, recombinant vaccinia viruses encoding full-length T antigen or epitope mingenes, and tumor cells expressing T antigen variants revealed that anti-PD-1 invigorates subdominant TCD8 responses by relieving their lysis-dependent suppression by immunodominant TCD8. Our work constitutes the first report that interfering with PD-1 signaling potentiates epitope spreading in tumor-specific responses, a finding with clear implications for cancer immunotherapy and vaccination.

For chronic viral infections like Hepatitis C, CD8-CTLs have emerged as important protective tools. Hence, isolated dominant epitopes arranged as polytope DNA or peptide vaccines represent a promising approach. However, because of controversial rules governing the polytope construction and epitope processing, proper design and primary analysis of such vaccines are prior to the costly transgenic animal studies. In this study, based on in silico epitope selection, four HLA-A2 (C132, E614 and N1406) and H-2d (E405 and C132) immunodominant CD8-epitopes of HCV were selected. The codon optimized nucleotide sequences of the epitopes were assembled by overlap extension PCR in three different sequential tandems for the best proteasomal cleavage predictions and cloned into pcDNA3.1 vector. In addition, to enhance particulate formation, three other plasmids containing the fusion of polytopes with hepatitis B surface antigen gene (HBsAg) were also constructed. Proper expression of all constructs in transfected Cos-7 cells was verified by RT-PCR, immunofluorescence, Western-blot, ELISA and dot blot techniques. Moreover, particle formation of HBsAg-fused polytopes was manifested by their secretion to the culture media albeit in lesser amounts compared to sole HBsAg protein. Finally, the positive delayed-type hypersensitivity (DTH) response of vaccinated mice indicated the in vivo expression of all constructs and efficient stimulation of immune response, which was stronger for HBsAg fusion constructs. In addition, proper processing of the epitopes was evidenced by the DTH response towards H-2d epitopic peptides. These data provide enough support and merit for the further evaluation of the designed constructs in HLA-A2 transgenic mice.