In this interview, Cerba Research describes how its teams were able to discover resident memory T cells in solid tumors thanks to multiplex immunofluorescence.
Can you begin by explaining how T cells function as effector cells and why this is important?
T cells are key effectors of immunity and are designated preferred targets for immune modulation. T cells They can be classified as either T effector (Teff) cells or T regulatory (Treg) cells.
Teff cells facilitate optimal immune responses against invading microbes and tumor antigens. Under homeostatic conditions, Tregs promote peripheral tolerance.
However, Tregs may inhibit Teff cell functions within tumors. The complex interactions between Teff and Treg determine the outcome of tumour-specific immune responses. Favorable survival in several cancers has been associated with elevated Teff-to-Treg cell ratios as determined by various methods such as flow cytometry or immunohistochemistry on serial sections.
Multiplex immunofluorescence confers a technical advantage by enabling detection of co-expression and spatial organization of multiple targets within a tissue architecture preserved on a single slide.
What is the main aim of your studies?
The main goal was to prove the usefulness of histalim histoprofil® -Trig Human and mouse multiplex immunofluorescence panel for identification of Treg and Teff cells On site.
What methods were used?
During the study, various methods were applied, including plate design, target validation, and a serial multiplexing protocol using OPAL.® (Akoya Biosciences) Fluorophores on BOND RX® (Leica) stained slide.
Multispectral images of patient subcutaneous tissue xenograft (PDX) model, human tissue microarrays, and non-small cell lung cancer (NSCLC) multispectral images were acquired using VECTRA.® PolarisTM slide scanner (Akoya Biosciences).
Could you explain how the board validation is done?
Plate validation is performed using a single- and multiplexed staining comparison. Simplex slides were stained for individual biomarkers in a simple protocol and compared to serial section stained with multiplex IHC.
Staining compatibility between the multiplex slide and the simplex slide was determined by running analysis with HALO® After multispectral deconvolution. The percentage of positive cells from the simplex slide was compared to the multiplex chip that yielded satisfactory results for both HISTOPROFILE® -Treg panels.
What are the results obtained during the study?
After applying Histoprofile®Assemble the human panel of three non-small cell lung cancer (NSCLC) tissues using a Leica Bond RX, multispectral images captured using a Vectra Polaris.
We disassembled the images obtained using INFORM softwareallowing us to detect CD3/CD8 double positive cells and Tregs (CD3+, CD4+, CD25+, FoxP3+) using the HALO module.
Teff/Treg variance between samples can easily be estimated.
How did the study inform your findings, and what was one of your conclusions during the study?
During the study, the approach demonstrates the effective power of the histoprofile® Multiplex Treg immunohistochemistry and how it best works to identify and quantify several immune cell populations on a single tissue. This reveals great application potential for this method across a wide range of tissues in clinical and preclinical settings.
Simplex slides were stained for each individual biomarker in a simple protocol and compared to serial section stained with HISTOPROFILE® TRM Multiplexing Panel Coloring concordance between the multiplexing slice and simple slices was determined by analysis using HALO® Without multispectral deconvolution a) Example image of a multiplex stained healthy human amygdala The co-expression of the five markers in panel CD103CD69CD8CD3CD49 a can be estimated at magnification b) Example images corresponding to a multiplex slice for CD8 (and simple slice) and the corresponding HALO® Masks indicate positive cells C) The percentage of positive cells from the simple (blue bars) and multiplex (purple bars) slides showing comparable staining profiles between slides. Protocol accuracy was determined by intra-run repeat analysis and between-run repeat analysis (data not shown). Image credit: Cerba Research
Image credit: Cerba Research
Three NSCLC samples were stained using HISTOPROFILE® TRM plate and photographed using VECTRA® PolarisTM full-scan multispectral images were analyzed using HALO® A) Example of a multiplex panel in each of the three NSCLC samples: CD8 (orange) CD103 (green) CD49a (white) CD69 (yellow) CD3 (red). Scale bar 40 μm In the literature, the TRM was modeled by the different combinations of markers used in the panel. The markers were combined to analyze different TRM CD8 subtypes+/ CD103, CD8+/CD49a, CD8+/ CD69, CD8+/ CD103+/ CD69, CD8+/ CD103+/ CD69+/CD49a in the neoplastic and non-neoplastic regions of the three human samples of NSCLC b) TYPICAL HISTOPROFILE IMAGES® TRM panel and individual groups with fluorescent image and cell masks corresponding to different TRM cell subtypes. Scale bar 100 µm C) Frequency of resident memory (CD8) T cells+ CD103+) in the tumoral region (blue bars) and the non-neoplastic region (purple bars) analyzed D) Normal distribution of different T-cell subpopulations residing in the tumoral and non-neoplastic regions of the three CD8 NSCLC samples+/ CD103+ (blue) CD8+/ CD49a+(purple)+/ CD69+(pale green), CD8+/ CD103+/ CD69+(rosy)+/ CD103+/ CD69+/ CD49a+(Dark blue). Image credit: Cerba Research
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