Detection of Dimers of Dimers of Human Leukocyte Antigen (HLA)–DR on the Surface of Living Cells by Single-Particle Fluorescence Imaging

The technique of single-particle fluorescence imaging was used to investigate the oligomeric state of MHC class II molecules on the surface of living cells. Cells transfected with human leukocyte antigen (HLA)–DR A and B genes were labeled at saturation with a univalent probe consisting of Fab coupled to R-phycoerythrin. Analysis of the intensities of fluorescent spots on the cell surface revealed the presence of single and double particles consistent with the simultaneous presence of HLA-DR heterodimers and dimers of dimers. The proportion of double particles was lower at 37°C than at 22°C, suggesting that the heterodimers and dimers of dimers exist in a temperature-dependent equilibrium. These results are discussed in the context of a possible role for HLA-DR dimers of dimers in T cell receptor–MHC interactions. The technique is validated by demonstrating that fluorescence imaging can distinguish between dimers and tetramers of human erythrocyte spectrin deposited from solution onto a solid substrate. The methodology will have broad applicability to investigation of the oligomeric state of immunological and other membrane-bound receptors in living cells.

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Measurements of associations of cell-surface receptors by single-particle fluorescence imaging

Biochemical Society Transactions ◽

10.1042/bst0311028 ◽

2003 ◽

Vol 31 (5) ◽

pp. 1028-1031 ◽

Cited By ~ 6

Author(s):

R.J. Cherry ◽

I.E.G. Morrison ◽

I. Karakikes ◽

R.E. Barber ◽

G. Silkstone ◽

...

Keyword(s):

Cell Surface ◽

Fluorescence Imaging ◽

Mhc Class Ii ◽

Single Particle ◽

Gaussian Function ◽

Human Leukocyte ◽

Cell Surface Receptors ◽

Intact Cells ◽

Surface Receptors ◽

Hla Dr

SPFI (single-particle fluorescence imaging) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labelled with small fluorescent particles. The images of particles are diffraction-limited spots that are analysed by fitting with a two-dimensional Gaussian function. The spot intensities depend on whether they arise from one or more particles; this provides the basis for determining self-association of cell-surface receptors. We have used this approach to determine dimerization of MHC class II molecules and its disruption by interface peptides. We have also exploited the positional information obtained from SPFI to detect co-localization of cell-surface molecules. This involves labelling two different molecules with different coloured fluorophores and determining their positions separately by dual wavelength imaging. The images are analysed to quantify the overlap of the particle images and hence determine the extent of co-localization of the labelled molecules. The technique provides quantification of the extent of co-localization and can detect whether co-localized molecules occur singly or in clusters. We have obtained preliminary data for co-localization of lipopolysaccharide and CD14 on intact cells. We also show that HLA-DR (human leukocyte antigen-DR) and CD74 are partially co-localized and that interaction between these molecules involves the peptide-binding groove of HLA-DR.

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Case Report: Interferon-γ Restores Monocytic Human Leukocyte Antigen Receptor (mHLA-DR) in Severe COVID-19 With Acquired Immunosuppression Syndrome

Frontiers in Immunology ◽

10.3389/fimmu.2021.645124 ◽

2021 ◽

Vol 12 ◽

Author(s):

Steffen Dickel ◽

Clemens Grimm ◽

Katharina Amschler ◽

Sebastian Uwe Schnitzler ◽

Julie Schanz ◽

...

Keyword(s):

Human Leukocyte Antigen ◽

Mhc Class Ii ◽

Adaptive Immune Response ◽

Human Leukocyte ◽

Clinical Effectiveness ◽

Interferon Γ ◽

Antigen Receptors ◽

Leukocyte Antigen ◽

Hla Dr ◽

Secondary Infections

BackgroundThe major histocompatibility complex (MHC) class II characterized by monocytes CD14+ expression of human leukocyte antigen receptors (HLA-DR), is essential for the synapse between innate and adaptive immune response in infectious disease. Its reduced expression is associated with a high risk of secondary infections in septic patients and can be safely corrected by Interferon-y (IFNy) injection. Coronavirus disease (COVID-19) induces an alteration of Interferon (IFN) genes expression potentially responsible for the observed low HLA-DR expression in circulating monocytes (mHLA-DR).MethodsWe report a case of one-time INFy injection (100 mcg s.c.) in a superinfected 61-year-old man with COVID-19–associated acute respiratory distress syndrome (ARDS), with monitoring of mHLA-DR expression and clinical tolerance.ObservationsLow mHLA-DR pretreatment expression (26.7%) was observed. IFNy therapy leading to a rapid increase in mHLA-DR expression (83.1%).ConclusionsSevere ARDS in a COVID-19 patient has a deep reduction in mHLA-DR expression concomitantly with secondary infections. The unique IFNy injection was safe and led to a sharp increase in the expression of mHLA-DR. Based on immune and infection monitoring, more cases of severe COVID-19 patients with low mHLA-DR should be treated by IFNy to test the clinical effectiveness.

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Regulation of Major Histocompatibility (MHC) Class II Human Leukocyte Antigen-DRα Gene Expression in Thyrocytes by Single Strand Binding Protein-1, a Transcription Factor That Also Regulates Thyrotropin Receptor and MHC Class I Gene Expression

Endocrinology ◽

10.1210/endo.139.5.5991 ◽

1998 ◽

Vol 139 (5) ◽

pp. 2300-2313 ◽

Cited By ~ 6

Author(s):

Pina L. Balducci-Silano ◽

Koichi Suzuki ◽

Masanori Ohta ◽

Jun Saito ◽

Masayuki Ohmori ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Human Leukocyte Antigen ◽

Mhc Class I ◽

Mhc Class Ii ◽

Human Leukocyte ◽

Thyrotropin Receptor ◽

Leukocyte Antigen ◽

I Gene ◽

Mhc Class I Gene

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Sampling From the Proteome to the Human Leukocyte Antigen-DR (HLA-DR) Ligandome ProceedsViaHigh Specificity

Molecular & Cellular Proteomics ◽

10.1074/mcp.m115.055780 ◽

2016 ◽

Vol 15 (4) ◽

pp. 1412-1423 ◽

Cited By ~ 38

Author(s):

Geert P. M. Mommen ◽

Fabio Marino ◽

Hugo D. Meiring ◽

Martien C. M. Poelen ◽

Jacqueline A. M. van Gaans-van den Brink ◽

...

Keyword(s):

Human Leukocyte Antigen ◽

Human Leukocyte ◽

Leukocyte Antigen ◽

Hla Dr

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Human Leukocyte Antigen (HLA)-Linked Control of Susceptibility to Pulmonary Tuberculosis and Association with HLA-DR Types

The Journal of Infectious Diseases ◽

10.1093/infdis/148.4.676 ◽

1983 ◽

Vol 148 (4) ◽

pp. 676-681 ◽

Cited By ~ 85

Author(s):

S. P. N. Singh ◽

N. K. Mehra ◽

H. B. Dingley ◽

J. N. Pande ◽

M. C. Vaidya

Keyword(s):

Pulmonary Tuberculosis ◽

Human Leukocyte Antigen ◽

Human Leukocyte ◽

Leukocyte Antigen ◽

Hla Dr

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Constitutive intracellular expression of human leukocyte antigen (HLA)-DO and HLA-DR but not HLA-DM in trophoblast cells

Human Immunology ◽

10.1016/j.humimm.2004.10.002 ◽

2005 ◽

Vol 66 (1) ◽

pp. 43-55 ◽

Cited By ~ 24

Author(s):

Anthi Ranella ◽

Simon Vassiliadis ◽

Chrisa Mastora ◽

Michailidou Valentina ◽

Eva Dionyssopoulou ◽

...

Keyword(s):

Human Leukocyte Antigen ◽

Human Leukocyte ◽

Trophoblast Cells ◽

Leukocyte Antigen ◽

Hla Dr ◽

Intracellular Expression

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Endogenous NGF regulates CGRP expression in human monocytes, and affects HLA-DR and CD86 expression and IL-10 production

Blood ◽

10.1182/blood-2004-10-4055 ◽

2005 ◽

Vol 106 (10) ◽

pp. 3507-3514 ◽

Cited By ~ 56

Author(s):

Luisa Bracci-Laudiero ◽

Luigi Aloe ◽

Maria Cristina Caroleo ◽

Pasquale Buanne ◽

Nicola Costa ◽

...

Keyword(s):

Mhc Class Ii ◽

T Cell Activation ◽

Cell Activation ◽

Human Leukocyte ◽

Interleukin 10 ◽

Human Monocytes ◽

Leukocyte Antigen ◽

Physiologic Mechanism ◽

Hla Dr ◽

Antigen Presenting

AbstractOur recent results on autocrine nerve growth factor (NGF) synthesis in B lymphocytes, which directly regulates the expression and release of calcitonin gene-related peptide (CGRP), a neuropeptide known to down-regulate immune response, led us to propose an anti-inflammatory action of NGF. In the present work, we investigated whether the endogenous synthesis of NGF can regulate the expression of CGRP in other antigen-presenting cells, such as monocytes, and whether this may have a functional effect. Our data indicate that human monocytes synthesize basal levels of NGF and CGRP and that, following lipopolysaccharide (LPS) stimulation, NGF and CGRP expression are both up-regulated. When endogenous NGF is neutralized, the up-regulation of CGRP expression induced by LPS is inhibited. The expression of membrane molecules involved in T-cell activation such as human leukocyte antigen-DR (HLA-DR) and CD86 is affected by endogenous NGF, and similar effects were obtained using a CGRP1 receptor antagonist. In addition, NGF deprivation in LPS-treated monocytes significantly decreases interleukin 10 (IL-10) synthesis. Our findings indicate that endogenous NGF synthesis has a functional role and may represent a physiologic mechanism to down-regulate major histocompatibility complex (MHC) class II and CD86 expression and alter the development of immune responses.

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Human Leukocyte Antigen-DQ8 Transgenic Mice: a Model To Examine the Toxicity of Aerosolized Staphylococcal Enterotoxin B

Infection and Immunity ◽

10.1128/iai.73.4.2452-2460.2005 ◽

2005 ◽

Vol 73 (4) ◽

pp. 2452-2460 ◽

Cited By ~ 38

Author(s):

Chad J. Roy ◽

Kelly L. Warfield ◽

Brent C. Welcher ◽

Raoul F. Gonzales ◽

Tom Larsen ◽

...

Keyword(s):

Human Leukocyte Antigen ◽

Mhc Class Ii ◽

Small Animal ◽

Human Leukocyte ◽

Class Ii ◽

Leukocyte Antigen ◽

Perivascular Edema ◽

Aerosol Exposure ◽

Mrna Gene ◽

Lethal Shock

ABSTRACT Staphylococcal enterotoxins (SEs) belong to a large group of bacterial exotoxins that cause severe immunopathologies, especially when delivered as an aerosol. SEs elicit the release of lethal amounts of cytokines by binding to major histocompatibility complex (MHC) class II and cross-linking susceptible T-cell receptors. Efforts to develop effective therapeutic strategies to protect against SEs delivered as an aerosol have been hampered by the lack of small animal models that consistently emulate human responses to these toxins. Here, we report that human leukocyte antigen-DQ8 (HLA-DQ8) transgenic (Tg) mice, but not littermate controls, succumbed to lethal shock induced by SEB aerosols without potentiation. Substantial amounts of perivascular edema and inflammatory infiltrates were noted in the lungs of Tg mice, similar to the pathology observed in nonhuman primates exposed by aerosol to SEB. Furthermore, the observed pathologies and lethal shock correlated with an upsurge in proinflammatory cytokine mRNA gene expression in the lungs and spleens, as well as with marked increases in the levels of proinflammatory circulating cytokines in the Tg mice. Unlike the case for littermate controls, telemetric evaluation showed significant hypothermia in Tg mice exposed to lethal doses of SEB. Taken together, these results show that this murine model will allow for the examination of therapeutics and vaccines developed specifically against SEB aerosol exposure and possibly other bacterial superantigens in the context of human MHC class II receptors.

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