When the Same Expression Profile Hides Different Immune States

A playbook on how protein organisation reveals functional differences beyond abundance

Figure legend: T cell activation is associated with alterations in the membrane protein organization.
Activation results in a profound reshaping of the surface landscape of T cells, including changes in the spatial organization of the proteins. Although critical for cellular function, the spatial organization of membrane proteins is typically overlooked by most traditional T cell analysis methods.

Scenario: When phenotype doesn’t explain function

You analyse the process of T cell activation using conventional flow cytometry - based protein
abundance profiling.

Comparing PHA - activated to resting T cells, you observe an increase in activation markers
such as CD69, CD25, and CD38 as expected.

Yet within these activated populations, functional heterogeneity remains:
✓ Cells with highly similar marker profiles behave differently
✓ Some cells acquire stronger effector function associated with activation
✓ While others show reduced function despite comparable expression patterns

A common interpretation

It is often assumed that:
“These cells express the same markers, therefore they are in the same functional
state.”

However, similar abundance profiles do not necessarily mean cells are biologically
equivalent.

As a result:
✓ Distinct functional states may be grouped together
✓ Regulatory mechanisms can remain hidden
✓ Biologically meaningful subsets may be overlooked

When to change approach

Consider expanding the analysis beyond abundance when:
✓ Functional differences persist despite similar expression profiles
✓ Known regulatory proteins show no abundance change
✓ Phenotyping alone does not explain the observed biology

The Missing Dimension: Protein Organisation

Cellular function is not only regulated by what proteins are expressed, but also by how these
proteins are arranged. This includes the formation of functional protein complexes, clusters of
proteins and exclusion of regulatory proteins.

Hence, to resolve hidden functional heterogeneity, analyse:
✓ Protein clustering
✓ Colocalisation
✓ Protein domains

What abundance alone does not show in activated T cells

Cell surface architecture analysis on resting and PHA - activated PBMCs, confirms upregulation
of activation markers CD69, CD25 and CD38.

On the other hand, CD82 abundance remains similar between resting and activated T cells.

But organisation analysis shows:
✓ Increased CD82 clustering
✓ Specifically within a subset of CD8 T cells
This subset is not identifiable from abundance measurements alone.

Figure legend: PHA - activated T cells reorganize CD82. (A) Abundance of CD82 in 3 donors, matched resting and activated samples. (B) Clustering of CD82 within the samples. (C) Clustering of CD82 is not dependent on CD82 abundance. (D) Cell visualizations sh owing unclustered CD82 in a resting cell (left) and clustered CD82 in an activated cell (right). (E) Confirmation of the CD82 clustering using fluorescent microscopy.

Why this matters

CD82 has previously been associated with functional responses and recruitment to the immune
synapse.

The observation here is not simply that CD82 is present, but that its membrane organisation
changes selectively within a subset of activated cells.

This suggests:
✓ Distinct functional states can exist within phenotypically similar populations
✓ Membrane organisation may reflect signalling readiness or functional potential

What the data supports

Directly shown
✓ Abundance defines activation - associated populations
✓ Organisation identifies additional cellular subsets invisible to abundance

Biological interpretation
✓ Protein organisation may contribute to functional regulation

Key concept

Protein abundance defines which proteins are present.
Protein organisation reveals how those proteins are arranged to function.

Takeaway

Cell surface architecture can reveal new cellular states.

Explore further about the technology and its application here:

Protein Interactomics by Proximity Networks - Pixelgen Technologies
Application note “Molecular Pixelation uncovers large - scale changes in the membrane protein architecture of activated T cells” link: https://www.pixelgen.com/application-notes/

GO BEYOND PROTEIN EXPRESSION

Measure more than which proteins are present. Map how cell surface proteins are organized, connected, and spatially arranged across single cells.

Pixelgen’s Proximity Network Assay brings nanoscale protein interactomics to high-throughput immune cell research, helping teams uncover biology that abundance-based methods can miss.

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