M2 Macrophage Suppression Assay

Macrophages - the most abundant antigen-presenting cells in the periphery - represent by themselves a wide group of functionally-different subpopulations involved in several immunological functions, including inflammatory responses and tissue homeostasis maintain & repair. Unlike M1 macrophages which promote a pro-inflammatory response, alternatively activated M2 macrophages, with diametrically opposed functions, trigger inflammation resolution and suppress T cell activation via several mechanisms including immune checkpoint engagement (e.g. PDL1), release of anti-inflammatory cytokines such as IL-10 and TGFb, and metabolic activities which promote essential amino acid depletion (e.g. Arginine). – which characteristics that they share with the so-called tumor-associated macrophages (TAMs).
 
Our newly validated M2 suppression assay based on i) the co-culture of autologous monocyte-derived M2 macrophages and activated CD4+ T cells (or PBMCs) and on ii) the quantitation of IFNg levels as surrogate of T cell activation, is specifically designed to assess new immunotherapeutics for their modulatory activity on the phenotype and function of M2 macrophages. Candidate compounds can thus be evaluated as single agents or in combinatorial treatments, for their potential to repolarize / switch M2 macrophages and to antagonize M2-mediated T cell suppression.

Illustrative data

Molecular features of human monocyte-derived macrophages through surface marker expression and cytokine profiles
While displaying an expected variation from a donor to another one, surface marker expression profiles (A) as well as their cytokine profiles (B) are maintained. Isolated human peripheral blood monocytes originating from two donors were subjected to M1 or M2 macrophage polarization process. At the end of the polarization process, CD80 and CD163 expression were assessed by flow cytometry thereby highlighting M1 macrophages as CD80High CD163Low and M2 macrophages as CD80Low and CD163High. Also, cytokine levels released in the supernatants were measured using a HTRF. M1 macrophages were shown to display a IL6High / IL12High / IL10Low profile while M2 display a IL6Low / IL12Low / IL10High profile.

While displaying an expected variation from a donor to another one, surface marker expression ...

Anti-inflammatory profile of M2 polarized macrophages is reversed upon p38 MAPK inhibitor treatment _______________
Treatment with a p38 MAPK inhibitor during the M2 polarization process is shown to be capable of repolarizing/switching M2 phenotype by reverting, at least partially but dose-dependently, the IL6low IL10high profile.

Treatment with a p38 MAPK inhibitor during the M2 polarization process is shown to be capable ...

T cell response immunosuppression mediated by M2 polarized macrophages is reversed by molecular pathway modulators and ICI
In the co-culture immunosuppression assay with PBMCs, M2 macrophages exhibit a strong suppression of T cell activity shown through released IFNg level decrease. M2 suppression function is known to be exerted via multiple mechanisms including PD1/PDL1 axis engagement or pathway modulation, among other ways. Interestingly, co-cultures of activated PBMC with M2 macrophages that underwent treatment with a p38 MAPK inhibitor during their polarization result, at least partially, in a dose-dependent reversal of the T cell activation suppression compared to untreated PBMC/M2 co-cultures. Atezolizumab treatment of co-cultures is also shown to completely and dose-dependently relieve the immunosuppressive M2 function on T cell response, with a reversal of the M2 IFNg release profile.

In the co-culture immunosuppression assay with PBMCs, M2 macrophages exhibit a strong suppression ...

For more information, download our macrophage suppression assay white paper!

Assay principle

M2 macrophage suppression assay










  • HTRF-based quantification of key cytokines released by macrophages, e.g. IL-6, IL-10, IL-12 as to evaluate the potency of candidate compounds to revert M2 phenotype.
  • Human autologous M2 suppression assay, based on a co-culture of monocyte-derived M2 macrophages with activated T cells (or PBMCs) and measurement of IFNγ, as one of the most representative surrogates of T cell activation.
  • Robust functional assay, microplate format tailored for screening campaigns
  • Complementary analysis can be implemented to decipher MOA (e.g. activation markers, signaling pathways, etc)