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1.
BMC Med ; 20(1): 353, 2022 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-36195867

RESUMO

BACKGROUND: Hormonal changes during the menstrual cycle play a key role in shaping immunity in the cervicovaginal tract. Cervicovaginal fluid contains cytokines, chemokines, immunoglobulins, and other immune mediators. Many studies have shown that the concentrations of these immune mediators change throughout the menstrual cycle, but the studies have often shown inconsistent results. Our understanding of immunological correlates of the menstrual cycle remains limited and could be improved by meta-analysis of the available evidence. METHODS: We performed a systematic review and meta-analysis of cervicovaginal immune mediator concentrations throughout the menstrual cycle using individual participant data. Study eligibility included strict definitions of the cycle phase (by progesterone or days since the last menstrual period) and no use of hormonal contraception or intrauterine devices. We performed random-effects meta-analyses using inverse-variance pooling to estimate concentration differences between the follicular and luteal phases. In addition, we performed a new laboratory study, measuring select immune mediators in cervicovaginal lavage samples. RESULTS: We screened 1570 abstracts and identified 71 eligible studies. We analyzed data from 31 studies, encompassing 39,589 concentration measurements of 77 immune mediators made on 2112 samples from 871 participants. Meta-analyses were performed on 53 immune mediators. Antibodies, CC-type chemokines, MMPs, IL-6, IL-16, IL-1RA, G-CSF, GNLY, and ICAM1 were lower in the luteal phase than the follicular phase. Only IL-1α, HBD-2, and HBD-3 were elevated in the luteal phase. There was minimal change between the phases for CXCL8, 9, and 10, interferons, TNF, SLPI, elafin, lysozyme, lactoferrin, and interleukins 1ß, 2, 10, 12, 13, and 17A. The GRADE strength of evidence was moderate to high for all immune mediators listed here. CONCLUSIONS: Despite the variability of cervicovaginal immune mediator measurements, our meta-analyses show clear and consistent changes during the menstrual cycle. Many immune mediators were lower in the luteal phase, including chemokines, antibodies, matrix metalloproteinases, and several interleukins. Only interleukin-1α and beta-defensins were higher in the luteal phase. These cyclical differences may have consequences for immunity, susceptibility to infection, and fertility. Our study emphasizes the need to control for the effect of the menstrual cycle on immune mediators in future studies.


Assuntos
Elafina , beta-Defensinas , Feminino , Fator Estimulador de Colônias de Granulócitos , Humanos , Imunoglobulinas , Fatores Imunológicos , Interferons , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-16 , Interleucina-1alfa , Interleucina-6 , Interleucinas , Lactoferrina , Ciclo Menstrual , Muramidase , Progesterona
2.
J Biol Chem ; 292(3): 802-813, 2017 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-27903649

RESUMO

T-cell cross-reactivity is essential for effective immune surveillance but has also been implicated as a pathway to autoimmunity. Previous studies have demonstrated that T-cell receptors (TCRs) that focus on a minimal motif within the peptide are able to facilitate a high level of T-cell cross-reactivity. However, the structural database shows that most TCRs exhibit less focused antigen binding involving contact with more peptide residues. To further explore the structural features that allow the clonally expressed TCR to functionally engage with multiple peptide-major histocompatibility complexes (pMHCs), we examined the ILA1 CD8+ T-cell clone that responds to a peptide sequence derived from human telomerase reverse transcriptase. The ILA1 TCR contacted its pMHC with a broad peptide binding footprint encompassing spatially distant peptide residues. Despite the lack of focused TCR-peptide binding, the ILA1 T-cell clone was still cross-reactive. Overall, the TCR-peptide contacts apparent in the structure correlated well with the level of degeneracy at different peptide positions. Thus, the ILA1 TCR was less tolerant of changes at peptide residues that were at, or adjacent to, key contact sites. This study provides new insights into the molecular mechanisms that control T-cell cross-reactivity with important implications for pathogen surveillance, autoimmunity, and transplant rejection.


Assuntos
Linfócitos T CD8-Positivos , Peptídeos , Receptores de Antígenos de Linfócitos T , Telomerase , Linfócitos T CD8-Positivos/química , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Reações Cruzadas , Humanos , Peptídeos/química , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/imunologia , Telomerase/química , Telomerase/imunologia
3.
J Immunol ; 194(10): 4814-24, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25862821

RESUMO

There is an urgent need for a better understanding of adaptive immunity to Burkholderia pseudomallei, the causative agent of melioidosis that is frequently associated with sepsis or death in patients in Southeast Asia and Northern Australia. The imperative to identify vaccine targets is driven both by the public health agenda in these regions and biological threat concerns. In several intracellular bacterial pathogens, alkyl hydroperoxidase reductases are upregulated as part of the response to host oxidative stress, and they can stimulate strong adaptive immunity. We show that alkyl hydroperoxidase reductase (AhpC) of B. pseudomallei is strongly immunogenic for T cells of 'humanized' HLA transgenic mice and seropositive human donors. Some T cell epitopes, such as p6, are able to bind diverse HLA class II heterodimers and stimulate strong T cell immunity in mice and humans. Importantly, patients with acute melioidosis who survive infection show stronger T cell responses to AhpC relative to those who do not. Although the sequence of AhpC is virtually invariant among global B. pseudomallei clinical isolates, a Cambodian isolate varies only in C-terminal truncation of the p6 T cell epitope, raising the possibility of selection by host immunity. This variant peptide is virtually unable to stimulate T cell immunity. For an infection in which there has been debate about centrality of T cell immunity in defense, these observations support a role for T cell immunity to AhpC in disease protection.


Assuntos
Burkholderia pseudomallei/genética , Burkholderia pseudomallei/imunologia , Melioidose/imunologia , Peroxirredoxinas/genética , Peroxirredoxinas/imunologia , Imunidade Adaptativa/imunologia , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Burkholderia pseudomallei/enzimologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Genótipo , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Camundongos , Camundongos Transgênicos
4.
Immunol Cell Biol ; 94(6): 573-82, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26846725

RESUMO

Evidence indicates that autoimmunity can be triggered by virus-specific CD8(+) T cells that crossreact with self-derived peptide epitopes presented on the cell surface by major histocompatibility complex class I (MHCI) molecules. Identification of the associated viral pathogens is challenging because individual T-cell receptors can potentially recognize up to a million different peptides. Here, we generate peptide length-matched combinatorial peptide library (CPL) scan data for a panel of virus-specific CD8(+) T-cell clones spanning different restriction elements and a range of epitope lengths. CPL scan data drove a protein database search limited to viruses that infect humans. Peptide sequences were ranked in order of likelihood of recognition. For all anti-viral CD8(+) T-cell clones examined in this study, the index peptide was either the top-ranked sequence or ranked as one of the most likely sequences to be recognized. Thus, we demonstrate that anti-viral CD8(+) T-cell clones are highly focused on their index peptide sequence and that 'CPL-driven database searching' can be used to identify the inciting virus-derived epitope for a given CD8(+) T-cell clone. Moreover, to augment access to CPL-driven database searching, we have created a publicly accessible webtool. Application of these methodologies in the clinical setting may clarify the role of viral pathogens in the etiology of autoimmune diseases.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/imunologia , Bases de Dados de Proteínas , HIV-1/imunologia , Herpesvirus Humano 4/imunologia , Humanos , Ligantes , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/metabolismo , Reprodutibilidade dos Testes , Especificidade da Espécie
5.
J Antimicrob Chemother ; 71(2): 372-86, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26514157

RESUMO

OBJECTIVES: The objectives of this study were to comprehensively assess mRNA expression of 84 drug transporters in human colorectal biopsies and six representative cell lines, and to investigate the alteration of drug transporter gene expression after exposure to three candidate microbicidal antiretroviral (ARV) drugs (tenofovir, darunavir and dapivirine) in the colorectal epithelium. The outcome of the objectives informs development of optimal ARV-based microbicidal formulations for prevention of HIV-1 infection. METHODS: Drug transporter mRNA expression was quantified from colorectal biopsies and cell lines by quantitative real-time PCR. Relative mRNA expression was quantified in Caco-2 cells and colorectal explants after induction with ARVs. Data were analysed using Pearson's product moment correlation (r), hierarchical clustering and principal component analysis (PCA). RESULTS: Expression of 58 of the 84 transporters was documented in colorectal biopsies, with genes for CNT2, P-glycoprotein (P-gp) and MRP3 showing the highest expression. No difference was noted between individual subjects when analysed by age, gender or anatomical site (rectum or recto-sigmoid) (r = 0.95-0.99). High expression of P-gp and CNT2 proteins was confirmed by immunohistochemical staining. Similarity between colorectal tissue and cell-line drug transporter gene expression was variable (r = 0.64-0.84). PCA showed distinct clustering of human colorectal biopsy samples, with the Caco-2 cells defined as the best surrogate system. Induction of Caco-2 cell lines with ARV drugs suggests that darunavir-based microbicides incorporating tenofovir may result in drug-drug interactions likely to affect distribution of individual drugs to sub-epithelial target cells. CONCLUSIONS: These findings will help optimize complex formulations of rectal microbicides to realize their full potential as an effective approach for pre-exposure prophylaxis against HIV-1 infection.


Assuntos
Anti-Infecciosos/metabolismo , Células Epiteliais/efeitos dos fármacos , Expressão Gênica , Mucosa Intestinal/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Idoso , Células CACO-2 , Darunavir/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Proteínas de Membrana Transportadoras/genética , Pessoa de Meia-Idade , Pirimidinas/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Tenofovir/metabolismo
6.
Blood ; 121(7): 1112-23, 2013 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-23255554

RESUMO

αß-TCRs expressed at the CD8(+) T-cell surface interact with short peptide fragments (p) bound to MHC class I molecules (pMHCI). The TCR/pMHCI interaction is pivotal in all aspects of CD8(+) T-cell immunity. However, the rules that govern the outcome of TCR/pMHCI engagement are not entirely understood, and this is a major barrier to understanding the requirements for both effective immunity and vaccination. In the present study, we discovered an unexpected feature of the TCR/pMHCI interaction by showing that any given TCR exhibits an explicit preference for a single MHCI-peptide length. Agonists of nonpreferred length were extremely rare, suboptimal, and often entirely distinct in sequence. Structural analysis indicated that alterations in peptide length have a major impact on antigenic complexity, to which individual TCRs are unable to adapt. This novel finding demonstrates that the outcome of TCR/pMHCI engagement is determined by peptide length in addition to the sequence identity of the MHCI-bound peptide. Accordingly, the effective recognition of pMHCI Ag, which is a prerequisite for successful CD8(+) T-cell immunity and protective vaccination, can only be achieved by length-matched Ag-specific CD8(+) T-cell clonotypes.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Sequência de Aminoácidos , Apresentação de Antígeno , Antígenos/química , Antígenos/genética , Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Clonais , Humanos , Imunidade Celular , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Fragmentos de Peptídeos/genética , Biblioteca de Peptídeos
7.
J Biol Chem ; 287(2): 1168-77, 2012 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-22102287

RESUMO

The T cell receptor (TCR) orchestrates immune responses by binding to foreign peptides presented at the cell surface in the context of major histocompatibility complex (MHC) molecules. Effective immunity requires that all possible foreign peptide-MHC molecules are recognized or risks leaving holes in immune coverage that pathogens could quickly evolve to exploit. It is unclear how a limited pool of <10(8) human TCRs can successfully provide immunity to the vast array of possible different peptides that could be produced from 20 proteogenic amino acids and presented by self-MHC molecules (>10(15) distinct peptide-MHCs). One possibility is that T cell immunity incorporates an extremely high level of receptor degeneracy, enabling each TCR to recognize multiple peptides. However, the extent of such TCR degeneracy has never been fully quantified. Here, we perform a comprehensive experimental and mathematical analysis to reveal that a single patient-derived autoimmune CD8(+) T cell clone of pathogenic relevance in human type I diabetes recognizes >one million distinct decamer peptides in the context of a single MHC class I molecule. A large number of peptides that acted as substantially better agonists than the wild-type "index" preproinsulin-derived peptide (ALWGPDPAAA) were identified. The RQFGPDFPTI peptide (sampled from >10(8) peptides) was >100-fold more potent than the index peptide despite differing from this sequence at 7 of 10 positions. Quantification of this previously unappreciated high level of CD8(+) T cell cross-reactivity represents an important step toward understanding the system requirements for adaptive immunity and highlights the enormous potential of TCR degeneracy to be the causative factor in autoimmune disease.


Assuntos
Autoimunidade , Linfócitos T CD8-Positivos/imunologia , Antígeno HLA-A2/imunologia , Insulina/imunologia , Modelos Imunológicos , Peptídeos/imunologia , Precursores de Proteínas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Diabetes Mellitus Tipo 1/imunologia , Humanos
8.
J Biol Chem ; 287(44): 37269-81, 2012 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-22952231

RESUMO

Altered peptide antigens that enhance T-cell immunogenicity have been used to improve peptide-based vaccination for a range of diseases. Although this strategy can prime T-cell responses of greater magnitude, the efficacy of constituent T-cell clonotypes within the primed population can be poor. To overcome this limitation, we isolated a CD8(+) T-cell clone (MEL5) with an enhanced ability to recognize the HLA A*0201-Melan A(27-35) (HLA A*0201-AAGIGILTV) antigen expressed on the surface of malignant melanoma cells. We used combinatorial peptide library screening to design an optimal peptide sequence that enhanced functional activation of the MEL5 clone, but not other CD8(+) T-cell clones that recognized HLA A*0201-AAGIGILTV poorly. Structural analysis revealed the potential for new contacts between the MEL5 T-cell receptor and the optimized peptide. Furthermore, the optimized peptide was able to prime CD8(+) T-cell populations in peripheral blood mononuclear cell isolates from multiple HLA A*0201(+) individuals that were capable of efficient HLA A*0201(+) melanoma cell destruction. This proof-of-concept study demonstrates that it is possible to design altered peptide antigens for the selection of superior T-cell clonotypes with enhanced antigen recognition properties.


Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/metabolismo , Antígeno HLA-A2/imunologia , Antígeno MART-1/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Apresentação de Antígeno , Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Dicroísmo Circular , Antígeno HLA-A2/química , Antígeno HLA-A2/metabolismo , Humanos , Cinética , Antígeno MART-1/química , Antígeno MART-1/metabolismo , Melanoma/imunologia , Melanoma/terapia , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Ressonância de Plasmônio de Superfície
9.
J Immunol ; 187(2): 654-63, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21677135

RESUMO

CD8(+) T cells recognize immunogenic peptides presented at the cell surface bound to MHCI molecules. Ag recognition involves the binding of both TCR and CD8 coreceptor to the same peptide-MHCI (pMHCI) ligand. Specificity is determined by the TCR, whereas CD8 mediates effects on Ag sensitivity. Anti-CD8 Abs have been used extensively to examine the role of CD8 in CD8(+) T cell activation. However, as previous studies have yielded conflicting results, it is unclear from the literature whether anti-CD8 Abs per se are capable of inducing effector function. In this article, we report on the ability of seven monoclonal anti-human CD8 Abs to activate six human CD8(+) T cell clones with a total of five different specificities. Six of seven anti-human CD8 Abs tested did not activate CD8(+) T cells. In contrast, one anti-human CD8 Ab, OKT8, induced effector function in all CD8(+) T cells examined. Moreover, OKT8 was found to enhance TCR/pMHCI on-rates and, as a consequence, could be used to improve pMHCI tetramer staining and the visualization of Ag-specific CD8(+) T cells. The anti-mouse CD8 Abs, CT-CD8a and CT-CD8b, also activated CD8(+) T cells despite opposing effects on pMHCI tetramer staining. The observed heterogeneity in the ability of anti-CD8 Abs to trigger T cell effector function provides an explanation for the apparent incongruity observed in previous studies and should be taken into consideration when interpreting results generated with these reagents. Furthermore, the ability of Ab-mediated CD8 engagement to deliver an activation signal underscores the importance of CD8 in CD8(+) T cell signaling.


Assuntos
Anticorpos/fisiologia , Antígenos CD8/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Citotoxicidade Imunológica , Antígenos HLA-A/química , Peptídeos/química , Receptores de Antígenos de Linfócitos T/deficiência , Anticorpos/metabolismo , Linfócitos T CD8-Positivos/citologia , Células Clonais , Antígenos HLA-A/imunologia , Antígeno HLA-A2 , Humanos , Imunofenotipagem , Ligantes , Peptídeos/análise , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/imunologia , Coloração e Rotulagem , Ressonância de Plasmônio de Superfície
10.
Front Immunol ; 13: 1029029, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36532063

RESUMO

Introduction: Immunological protection against human immunodeficiency virus-1 (HIV-1) infection is likely to require both humoral and cell-mediated immune responses, the latter involving cytotoxic CD8 T-cells. Characterisation of CD8 T-cell mediated direct anti-viral activity would provide understanding of potential correlates of immune protection and identification of critical epitopes associated with HIV-1 control. Methods: The present report describes a functional viral inhibition assay (VIA) to assess CD8 T-cell-mediated inhibition of replication of a large and diverse panel of 45 HIV-1 infectious molecular clones (IMC) engineered with a Renilla reniformis luciferase reporter gene (LucR), referred to as IMC-LucR. HIV-1 IMC replication in CD4 T-cells and CD8 T-cell mediated inhibition was characterised in both ART naive subjects living with HIV-1 covering a broad human leukocyte antigen (HLA) distribution and compared with uninfected subjects. Results & discussion: CD4 and CD8 T-cell lines were established from subjects vaccinated with a candidate HIV-1 vaccine and provided standard positive controls for both assay quality control and facilitating training and technology transfer. The assay was successfully established across 3 clinical research centres in Kenya, Uganda and the United Kingdom and shown to be reproducible. This IMC-LucR VIA enables characterisation of functional CD8 T-cell responses providing a tool for rational T-cell immunogen design of HIV-1 vaccine candidates and evaluation of vaccine-induced T-cell responses in HIV-1 clinical trials.


Assuntos
Infecções por HIV , HIV-1 , Humanos , Linfócitos T CD8-Positivos , Luciferases , Células Clonais
11.
AIDS ; 35(8): 1167-1177, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33710028

RESUMO

OBJECTIVES: Interleukin-21 (IL-21) has been linked with the generation of virus-specific memory CD8+ T cells following acute infection with HIV-1 and reduced exhaustion of CD8+ T cells. IL-21 has also been implicated in the promotion of CD8+ T-cell effector functions during viral infection. Little is known about the expression of interleukin-21 receptor (IL-21R) during HIV-1 infection or its role in HIV-1-specific CD8+ T-cell maintenance and subsequent viral control. METHODS: We compared levels of IL-21R expression on total and memory subsets of CD8+ T cells from HIV-1-negative and HIV-1-positive donors. We also measured IL-21R on antigen-specific CD8+ T cells in volunteers who were positive for HIV-1 and had cytomegalovirus-responding T cells. Finally, we quantified plasma IL-21 in treatment-naive HIV-1-positive individuals and compared this with IL-21R expression. RESULTS: IL-21R expression was significantly higher on CD8+ T cells (P = 0.0256), and on central memory (P = 0.0055) and effector memory (P = 0.0487) CD8+ T-cell subsets from HIV-1-positive individuals relative to HIV-1-negative individuals. For those infected with HIV-1, the levels of IL-21R expression on HIV-1-specific CD8+ T cells correlated significantly with visit viral load (r = 0.6667, P = 0.0152, n = 13) and inversely correlated with plasma IL-21 (r = -0.6273, P = 0.0440, n = 11). Lastly, CD8+ T cells from individuals with lower set point viral load who demonstrated better viral control had the lowest levels of IL-21R expression and highest levels of plasma IL-21. CONCLUSION: Our data demonstrates significant associations between IL-21R expression on peripheral CD8+ T cells and viral load, as well as disease trajectory. This suggests that the IL-21 receptor could be a novel marker of CD8+ T-cell dysfunction during HIV-1 infection.


Assuntos
Infecções por HIV , HIV-1 , Linfócitos T CD8-Positivos , Humanos , Subunidade alfa de Receptor de Interleucina-21 , Receptores de Interleucina-21 , Carga Viral
12.
PLoS One ; 16(11): e0260118, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34788349

RESUMO

Full characterisation of functional HIV-1-specific T-cell responses, including identification of recognised epitopes linked with functional antiviral responses, would aid development of effective vaccines but is hampered by HIV-1 sequence diversity. Typical approaches to identify T-cell epitopes utilising extensive peptide sets require subjects' cell numbers that exceed feasible sample volumes. To address this, CD8 T-cells were polyclonally expanded from PBMC from 13 anti-retroviral naïve subjects living with HIV using CD3/CD4 bi-specific antibody. Assessment of recognition of individual peptides within a set of 1408 HIV-1 Gag, Nef, Pol and Env potential T-cell epitope peptides was achieved by sequential IFNγ ELISpot assays using peptides pooled in 3-D matrices followed by confirmation with single peptides. A Renilla reniformis luciferase viral inhibition assay assessed CD8 T-cell-mediated inhibition of replication of a cross-clade panel of 10 HIV-1 isolates, including 9 transmitted-founder isolates. Polyclonal expansion from one frozen PBMC vial provided sufficient CD8 T-cells for both ELISpot steps in 12 of 13 subjects. A median of 33 peptides in 16 epitope regions were recognised including peptides located in previously characterised HIV-1 epitope-rich regions. There was no significant difference between ELISpot magnitudes for in vitro expanded CD8 T-cells and CD8 T-cells directly isolated from PBMCs. CD8 T-cells from all subjects inhibited a median of 7 HIV-1 isolates (range 4 to 10). The breadth of CD8 T-cell mediated HIV-1 inhibition was significantly positively correlated with CD8 T-cell breadth of peptide recognition. Polyclonal CD8 T-cell expansion allowed identification of HIV-1 isolates inhibited and peptides recognised within a large peptide set spanning the major HIV-1 proteins. This approach overcomes limitations associated with obtaining sufficient cell numbers to fully characterise HIV-1-specific CD8 T-cell responses by different functional readouts within the context of extreme HIV-1 diversity. Such an approach will have useful applications in clinical development for HIV-1 and other diseases.


Assuntos
Epitopos de Linfócito T , HIV-1 , Linfócitos T CD8-Positivos , Soropositividade para HIV , Inibição Psicológica , Leucócitos Mononucleares
13.
Front Immunol ; 12: 634832, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33777023

RESUMO

Individuals infected with HIV display varying rates of viral control and disease progression, with a small percentage of individuals being able to spontaneously control infection in the absence of treatment. In attempting to define the correlates associated with natural protection against HIV, extreme heterogeneity in the datasets generated from systems methodologies can be further complicated by the inherent variability encountered at the population, individual, cellular and molecular levels. Furthermore, such studies have been limited by the paucity of well-characterised samples and linked epidemiological data, including duration of infection and clinical outcomes. To address this, we selected 10 volunteers who rapidly and persistently controlled HIV, and 10 volunteers each, from two control groups who failed to control (based on set point viral loads) from an acute and early HIV prospective cohort from East and Southern Africa. A propensity score matching approach was applied to control for the influence of five factors (age, risk group, virus subtype, gender, and country) known to influence disease progression on causal observations. Fifty-two plasma proteins were assessed at two timepoints in the 1st year of infection. We independently confirmed factors known to influence disease progression such as the B*57 HLA Class I allele, and infecting virus Subtype. We demonstrated associations between circulating levels of MIP-1α and IL-17C, and the ability to control infection. IL-17C has not been described previously within the context of HIV control, making it an interesting target for future studies to understand HIV infection and transmission. An in-depth systems analysis is now underway to fully characterise host, viral and immunological factors contributing to control.


Assuntos
Infecções por HIV/diagnóstico , HIV-1/crescimento & desenvolvimento , Replicação Viral , Proteínas Adaptadoras de Transdução de Sinal/sangue , Adulto , África , Biomarcadores/sangue , Progressão da Doença , Feminino , Infecções por HIV/sangue , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/patogenicidade , Antígenos HLA-B/genética , Antígenos HLA-B/imunologia , Humanos , Incidência , Interleucina-17/sangue , Masculino , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Fatores Sexuais , Fatores de Tempo , Carga Viral , Adulto Jovem
14.
Am J Reprod Immunol ; 79(2)2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29286179

RESUMO

PROBLEM: Fluctuating hormones regulate reproductive processes in the female genital tract. Consequent changes in the local immunological environment are likely to affect cellular interaction with infectious agents and the assessment of therapies that target mucosal infections. METHOD OF STUDY: We compared Softcup and Weck-Cel sampling protocols and assessed the changes in the concentrations of 39 soluble proteins with menstrual cycle progression in the mucosal and peripheral compartments. RESULTS: We demonstrate that the mucosal immunological profile is distinct from serum with inflammatory and migratory signatures that are localized throughout the cycle. The analytes highlighted in the mucosal compartment were generally highest at the follicular phase with a tendency to fall as the cycle progressed through ovulation to the luteal phase. CONCLUSION: Our results underscore the need to consider these localized cyclical differences in studies aimed at assessing the outcome of disease and the efficacy of mucosal vaccines and other therapies.


Assuntos
Genitália Feminina/imunologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Imunoproteínas/metabolismo , Ciclo Menstrual/imunologia , Mucosa/imunologia , Periodicidade , Adolescente , Adulto , Boranos/metabolismo , Feminino , Hormônios Esteroides Gonadais/metabolismo , Humanos , Pessoa de Meia-Idade , Adulto Jovem
15.
Sci Immunol ; 3(29)2018 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-30413420

RESUMO

Killer cell immunoglobulin-like receptors (KIRs) are expressed predominantly on natural killer cells, where they play a key role in the regulation of innate immune responses. Recent studies show that inhibitory KIRs can also affect adaptive T cell-mediated immunity. In mice and in human T cells in vitro, inhibitory KIR ligation enhanced CD8+ T cell survival. To investigate the clinical relevance of these observations, we conducted an extensive immunogenetic analysis of multiple independent cohorts of HIV-1-, hepatitis C virus (HCV)-, and human T cell leukemia virus type 1 (HTLV-1)-infected individuals in conjunction with in vitro assays of T cell survival, analysis of ex vivo KIR expression, and mathematical modeling of host-virus dynamics. Our data suggest that functional engagement of inhibitory KIRs enhances the CD8+ T cell response against HIV-1, HCV, and HTLV-1 and is a significant determinant of clinical outcome in all three viral infections.


Assuntos
Linfócitos T CD8-Positivos/imunologia , HIV-1/imunologia , Hepacivirus/imunologia , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Receptores KIR/imunologia , Humanos
16.
J Clin Invest ; 128(4): 1569-1580, 2018 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-29528337

RESUMO

Polypeptide vaccines effectively activate human T cells but suffer from poor biological stability, which confines both transport logistics and in vivo therapeutic activity. Synthetic biology has the potential to address these limitations through the generation of highly stable antigenic "mimics" using subunits that do not exist in the natural world. We developed a platform based on D-amino acid combinatorial chemistry and used this platform to reverse engineer a fully artificial CD8+ T cell agonist that mirrored the immunogenicity profile of a native epitope blueprint from influenza virus. This nonnatural peptide was highly stable in human serum and gastric acid, reflecting an intrinsic resistance to physical and enzymatic degradation. In vitro, the synthetic agonist stimulated and expanded an archetypal repertoire of polyfunctional human influenza virus-specific CD8+ T cells. In vivo, specific responses were elicited in naive humanized mice by subcutaneous vaccination, conferring protection from subsequent lethal influenza challenge. Moreover, the synthetic agonist was immunogenic after oral administration. This proof-of-concept study highlights the power of synthetic biology to expand the horizons of vaccine design and therapeutic delivery.


Assuntos
Materiais Biomiméticos , Vírus da Influenza A/imunologia , Vacinas contra Influenza , Infecções por Orthomyxoviridae , Biblioteca de Peptídeos , Vacinação , Animais , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Células Cultivadas , Humanos , Vacinas contra Influenza/química , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/farmacologia , Camundongos , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/prevenção & controle
17.
J Immunol Methods ; 433: 6-16, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26921630

RESUMO

Multiplex bead-based assays have many advantages over ELISA, particularly for the analyses of large quantities of samples and/or precious samples of limited volume. Although many commercial arrays covering multitudes of biologically significant analytes are available, occasionally the development of custom arrays is necessary. Here, the development of a custom pentaplex sandwich immunoassay using Protein G-coupled beads, for analysis using the Luminex® xMAP® platform, is described. This array was required for the measurement of candidate biomarkers of vaccine safety in small volumes of mouse sera. Optimisation of this assay required a stepwise approach: testing cross-reactivity of the antibody pairs, the development of an in-house serum diluent buffer as well as heat-inactivation of serum samples to prevent interference from matrix effects. We then demonstrate the use of this array to analyse inflammatory mediators in mouse serum after immunisation. The work described here exemplifies how Protein G-coupled beads offer a flexible and robust approach to develop custom multiplex immunoassays, which can be applied to a range of analytes from multiple species.


Assuntos
Anticorpos/análise , Proteínas de Bactérias/química , Imunoensaio/métodos , Vacinas contra Influenza/administração & dosagem , Microesferas , Animais , Biomarcadores , Proteína C-Reativa/imunologia , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Feminino , Vacinas contra Influenza/efeitos adversos , Glicoproteínas de Membrana/imunologia , Camundongos , Proteínas do Tecido Nervoso/imunologia , Receptores Imunológicos/imunologia , Receptor Gatilho 1 Expresso em Células Mieloides
18.
Sci Rep ; 6: 35332, 2016 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-27748447

RESUMO

CD8+ T-cells play a role in the pathogenesis of autoimmune diseases such as multiple sclerosis and type 1 diabetes. However, drugs that target the entire CD8+ T-cell population are not desirable because the associated lack of specificity can lead to unwanted consequences, most notably an enhanced susceptibility to infection. Here, we show that autoreactive CD8+ T-cells are highly dependent on CD8 for ligand-induced activation via the T-cell receptor (TCR). In contrast, pathogen-specific CD8+ T-cells are relatively CD8-independent. These generic differences relate to an intrinsic dichotomy that segregates self-derived and exogenous antigen-specific TCRs according to the monomeric interaction affinity with cognate peptide-major histocompatibility complex class I (pMHCI). As a consequence, "blocking" anti-CD8 antibodies can suppress autoreactive CD8+ T-cell activation in a relatively selective manner. These findings provide a rational basis for the development and in vivo assessment of novel therapeutic strategies that preferentially target disease-relevant autoimmune responses within the CD8+ T-cell compartment.


Assuntos
Anticorpos/imunologia , Linfócitos T CD8-Positivos/citologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Doenças Autoimunes/imunologia , Antígenos CD8/imunologia , Linhagem Celular , Epitopos/metabolismo , Humanos , Terapia de Imunossupressão , Ilhotas Pancreáticas/metabolismo , Ligantes , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Peptídeos/metabolismo
19.
PLoS One ; 10(6): e0131405, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26102284

RESUMO

Anti-retroviral (ARV) -based microbicides are one of the strategies pursued to prevent HIV-1 transmission. Delivery of ARV drugs to subepithelial CD4+ T cells at concentrations for protection is likely determined by drug transporters expressed in the cervicovaginal epithelium. To define the role of drug transporters in mucosal disposition of topically applied ARV-based microbicides, these must be tested in epithelial cell line-based biopharmaceutical assays factoring the effect of relevant drug transporters. We have characterised gene expression of influx and efflux drug transporters in a panel of cervicovaginal cell lines and compared this to expression in cervicovaginal tissue. We also investigated the effect of dapivirine, darunavir and tenofovir, currently at advanced stages of microbicides development, on expression of drug transporters in cell lines. Expression of efflux ABC transporters in cervical tissue was best represented in HeLa, Ect1/E6E7 and End1/E6E7 cell lines. Expression of influx OCT and ENT transporters in ectocervix matched expression in Hela while expression of influx SLCO transporters in vagina was best reflected in VK2/E6E7 cell line. Stimulation with darunavir and dapivirine upregulated MRP transporters, including MRP5 involved in transport of tenofovir. Dapivirine also significantly downregulated tenofovir substrate MRP4 in cervical cell lines. Treatment with darunavir and dapivirine showed no significant effect on expression of BCRP, MRP2 and P-glycoprotein implicated in efflux of different ARV drugs. Darunavir strongly induced expression in most cell lines of CNT3 involved in cell uptake of nucleotide/nucleoside analogue reverse transcriptase inhibitors and SLCO drug transporters involved in cell uptake of protease inhibitors. This study provides insight into the suitability of cervicovaginal cell lines for assessment of ARV drugs in transport kinetics studies. The modulatory effect of darunavir and dapivirine on expression of drug transporters involved in transport of tenofovir points to the possibility of combining these drugs to improve retention of individual drugs at target tissues.


Assuntos
Fármacos Anti-HIV/farmacologia , Colo do Útero/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana Transportadoras/biossíntese , Vagina/efeitos dos fármacos , Adulto , Transporte Biológico , Linhagem Celular , Linhagem Celular Tumoral , Colo do Útero/citologia , Colo do Útero/metabolismo , Darunavir/farmacologia , Resistência a Medicamentos , Sinergismo Farmacológico , Feminino , Humanos , Proteínas de Membrana Transportadoras/genética , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Pirimidinas/farmacologia , Tenofovir/farmacologia , Vagina/citologia , Vagina/metabolismo
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