Phenotypic characterization of the tumor microenvironment during imiquimod treatment of melanoma in situ, lentiginous type

Follicular helper CD4 T (Tfh) cells play an essential role in the formation of germinal centers (GCs), where mature B cells proliferate, differentiate, and provide long-term protective humoral responses. Despite the extensive phenotypic characterization and identification of human Tfh cell subsets, their spatial positioning at tissue level is not well understood. Here, we describe a quantitative multiplexed immunofluorescence approach allowing for the comprehensive in situ characterization of Tfh cells in human tonsils and lymph nodes (LNs) from individuals with angioimmunoblastic T-cell lymphoma (AITL). We have developed eight multiplexed panels comprising a spectrum of Tfh cell markers, like PD-1, CXCR5, and ICOS, along with transcription factors (Bcl6, Tbet, GATA3), to assess their expression, frequencies, spatial distribution and co-localization in a quantitative manner. Combined analysis of relevant markers revealed the presence of several Tfh cell subsets at tissue level based on the differential expression of surface receptors, nuclear factors as well as their distinct localization within the follicular areas. Interestingly, we found a considerable amount of tonsillar Tfh cells expressing high levels of the Th2 regulator GATA3. The co-expression of GATA3, CXCR5, and BCL6, points to an important role of GATA3 for the generation of effector human Tfh cells. Furthermore, our data revealed significantly different Tfh cell profile signatures between health and disease. Therefore, our imaging platform generates meaningful information for the in situ characterization of human Tfh cells and could provide the base for future studies aiming to a comprehensive understanding of Tfh cell tissue heterogeneity.

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CD8+ T Lymphocytes Immune Depletion and LAG-3 Overexpression in Hodgkin Lymphoma Tumor Microenvironment Exposed to Anti-PD-1 Immunotherapy

Cancers ◽

10.3390/cancers13215487 ◽

2021 ◽

Vol 13 (21) ◽

pp. 5487

Author(s):

Jean-Marie Michot ◽

Severine Mouraud ◽

Julien Adam ◽

Julien Lazarovici ◽

Camille Bigenwald ◽

...

Keyword(s):

Pilot Study ◽

Tumor Microenvironment ◽

T Lymphocytes ◽

Hodgkin Lymphoma ◽

Phenotypic Characterization ◽

Cell Surfaces ◽

Cd8 T Lymphocytes ◽

Tumor Tissues ◽

Before And After

Background: Resistance to anti-PD-1 remains a considerable clinical challenge for the treatment of patients with classical Hodgkin lymphoma (cHL), and mechanisms of anti-PD-1 resistance remain unknown. This pilot study aims to investigate the tumor microenvironment in patients with cHL relapsing after anti-PD-1. Methods: This study investigated tumor samples of eight patients with cHL, including four patients exposed to anti-PD-1 with a paired longitudinal histological analysis before and after anti-PD-1, and four patients not exposed to anti-PD-1 who served as control for the cellular biological investigations. Fresh cells tumor microenvironment analysis included phenotypic characterization of their T cell surfaces immune checkpoint markers PD-1, PD-L1, ICOS, TIM-3, LAG-3, 41-BB and BTLA. Tumor tissues immunohistochemistry staining included CD30, CD4, CD8, CD68, CD163, PD-L1, PD-1, LAG-3 and TIM-3. Findings: Paired longitudinal tumor tissues analysis in the tumor microenvironment found a CD8+ lymphocytes tumor depletion and an increase of LAG-3 staining after anti-PD-1 exposure. The fresh cells analysis of the tumor microenvironment in patients exposed to anti-PD-1 found CD8+ lymphocyte depletion, with an elevated CD4+/CD8+ lymphocytes ratio (median ratio 9.77 in exposed anti-PD-1 versus 2.39 in not-exposed anti-PD-1 patients; p = 0.0943). On the cell surfaces of CD4+ lymphocytes, the median positive expression of LAG-3 was significantly higher in the samples exposed to anti-PD-1 compared to the controls (15.05 [IQR:17.91–10.65] versus 3.84 [IQR 1.87–6.57]; p = 0.0376). Interpretation: This pilot study proposes hypotheses for understanding the resistance to immunotherapies in patients with Hodgkin lymphoma. Hodgkin lymphoma exposed to anti-PD-1 correlated in tumor microenvironment with an immune depletion of CD8+ T lymphocytes and overexpression of LAG-3 on CD4+ helper T lymphocytes.

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Combining In Situ Hybridization with Retrograde Tracing and Immunohistochemistry for Phenotypic Characterization of Individual Neurons

Drugs of Abuse ◽

10.1385/1-59259-358-5:137 ◽

2003 ◽

pp. 137-152 ◽

Cited By ~ 1

Author(s):

Catherine Le Moine

Keyword(s):

In Situ Hybridization ◽

Retrograde Tracing ◽

Phenotypic Characterization

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Immunohistochemical characterization of benign activation of junctional melanocytes and melanoma in situ of the nail unit

Journal of Cutaneous Pathology ◽

10.1111/cup.13457 ◽

2019 ◽

Vol 46 (7) ◽

pp. 479-483 ◽

Cited By ~ 1

Author(s):

Alex Chu ◽

Josette André ◽

Phoebe Rich ◽

Sancy Leachman ◽

Curtis T. Thompson

Keyword(s):

Melanoma In Situ

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The Use of Advanced Analytical Techniques for Characterization of the Chemical, Physical, and Crystallographic Nature of a Surface

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100071107 ◽

1973 ◽

Vol 31 ◽

pp. 132-133 ◽

Cited By ~ 1

Author(s):

R. E. Herfert

Keyword(s):

Surface Characterization ◽

Analytical Techniques ◽

X Ray Diffraction ◽

Depth Of Penetration ◽

X Ray ◽

The Past ◽

Transmission Electron ◽

Scanning Electron

Studies of the nature of a surface, either metallic or nonmetallic, in the past, have been limited to the instrumentation available for these measurements. In the past, optical microscopy, replica transmission electron microscopy, electron or X-ray diffraction and optical or X-ray spectroscopy have provided the means of surface characterization. Actually, some of these techniques are not purely surface; the depth of penetration may be a few thousands of an inch. Within the last five years, instrumentation has been made available which now makes it practical for use to study the outer few 100A of layers and characterize it completely from a chemical, physical, and crystallographic standpoint. The scanning electron microscope (SEM) provides a means of viewing the surface of a material in situ to magnifications as high as 250,000X.

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A very rapid in situ Kikuchi technique to determine relative crystal misorientation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100106211 ◽

1988 ◽

Vol 46 ◽

pp. 830-831

Author(s):

J. I. Bennetch

Keyword(s):

Electron Beam ◽

Analytical Techniques ◽

Superplastic Forming ◽

The Other ◽

Kikuchi Pattern ◽

Kikuchi Line ◽

Line Analysis

In a recent study of the superplastic forming (SPF) behavior of certain Al-Li-X alloys, the relative misorientation between adjacent (sub)grains proved to be an important parameter. It is well established that the most accurate way to determine misorientation across boundaries is by Kikuchi line analysis. However, the SPF study required the characterization of a large number of (sub)grains in each sample to be statistically meaningful, a very time-consuming task even for comparatively rapid Kikuchi analytical techniques.In order to circumvent this problem, an alternate, even more rapid in-situ Kikuchi technique was devised, eliminating the need for the developing of negatives and any subsequent measurements on photographic plates. All that is required is a double tilt low backlash goniometer capable of tilting ± 45° in one axis and ± 30° in the other axis. The procedure is as follows. While viewing the microscope screen, one merely tilts the specimen until a standard recognizable reference Kikuchi pattern is centered, making sure, at the same time, that the focused electron beam remains on the (sub)grain in question.

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