T regulatory cells – many ways how to inspect this population

Jiri Hrdy is a head of immunology at Institute of Immunology and Microbiology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic.

"I have been interested in priming of immature neonatal immune since my master study. During development of newborn´s immune system, both effector and immunoregulatory responses have to be set. This led me to focus on T regulatory cells (Tregs)."

Tregs are in the center of research interest for many reasons resulting from their both physiological and pathological roles. Their main function is to suppress inadequate immune responses either to autoantigens or relatively innocuous environmental antigens such as pollen, dust, mites, antigens present in food or compounds of our microbiota. You can easily imagine a broad spectra of diseases related to impaired function of Tregs.

On the other hand, increased number and function of Tregs may be linked to certain disorders (e.g. tumor, autoimmune diseases) or insufficient anti-infectious immunity. In the case of anti-infectious immunity, pathogens (e.g. viruses) are able to induce Tregs limiting host immune responses against pathogens. Therefore, Tregs could serve as a double edged sword.

Importantly, not only the proportion but also the function of Tregs should be inspected. The main functions of Tregs include limitation of proliferation of effector cells by capturing IL-2 via high affinity receptor for IL-2 present on Tregs (CD25). The second function is mediated distinct cell surface molecules with regulatory function present on Treg (e.g. CTLA4, GITR, GARP, LAG3, CD39, CD73, CD101, PD-1/PD1L). Some modes of actions are dependent on cell to cell contact. The third option how Tregs promote suppression of immune responses is based on release of cytokines with regulatory functions (IL-10, IL-35, TGF-β).

Detection of Treg is not an easy task due to their substantial heterogeneity. Two main groups of Tregs are generally acknowledged; natural Tregs (nTregs) and induced Tregs (iTregs). nTregs arise in thymus and their T cell receptor (TCR) can recognize autoantigens. nTregs are involved in maintenance of central tolerance. nTregs can be defined as CD4⁺CD25^highCD127^lowFoxP3⁺Helios⁺ where FoxP3 and Helios are transcription markers present in nucleus therefore techniques including intracellular staining must be applied. Marker CD127 is applicable only for Treg analyses in clinical trials or cell cultures using cells of human origin.

The second group, iTregs, consists of CD4⁺CD25^highCD127^lowFoxP3⁺Helios⁺ subpopulation, Tr1 and Th3. Tr1 subpopulation was originally defined as CD4⁺IL-10⁺ but now other markers (CD49d, LAG3) enable us better determination of this population. Tr1 is considered to play a role on specific allergen immunotherapy. Th3 is known for its production of TGF-β. Recently, other Treg subpopulations have been described but their relationship to other Treg subpopulations and their role needs to be better clarified.

Selection of suitable reagents for proper Treg detection is critical. Distinct clones of antibodies against FoxP3 in combination with Treg detection kits can give substantially different results. Some clones have the superior capacity to bind to FoxP3 which can be supported by the usage of appropriate kit for staining of transcription factors. In our study, proportion and functional characteristics of Tregs in cord blood of newborns of healthy and allergic mothers were compared. We hypothesized that impaired immunoregulation apparent already on the level of cord blood could predispose children for future allergy development. To determine Tregs, we used TregFlowEx Kit manufactured by EXBIO.

TregFlowEx Kit contains reagents for intracellular staining of transcription factor. Moreover, antibodies against CD4, CD25 and FoxP3 are already included in TregFlowEx Kit. To better distinguish Treg subpopulations, additional markers were used: CD127 and Helios not included in the kit. Results are affected by the number of markers used as shown below. Combination of only two markers (CD4, CD25) initially used for Treg detection gives us the highest percentages of Treg (Figure 1) in contrast to group of markers targeting both cell surface and intracellular markers (CD4, CD25, CD127, FoxP3, Figure 4, and/or Helios, Figure 5). Differences can arise from distinct gating strategy and combination of markers used, Figure 5A and Figure 5B.

Download FIGURES 1-5 here.

In conclusion, careful selection of markers characterizing Treg is critical. In addition to that, usage of suitable kit for intracellular staining of transcription factor is important and partly depends on sample (whole peripheral blood versus cell homogenate from tissue, etc.). Notably, difference in literature on Treg proportion could be at least in part explained by the usage of distinct clones of antibodies against FoxP3, different kits for intracellular staining, gating strategies and number of markers used for Treg characterization. Therefore, proper staining procedure with clear gating strategy must accompany all the paper reporting on Tregs.

Recently published papers:
Lactobacillus reuteri 5454 and Bifidobacterium animalis ssp. lactis 5764 improve colitis while differentially impacting dendritic cells maturation and antimicrobial responses
In Vitro Characterization of Gut Microbiota-Derived Commensal Strains: Selection of Parabacteroides distasonis Strains Alleviating TNBS-Induced Colitis in Mice
Different immune response of dendritic cells of newborns of allergic and healthy mothers to bacterial stimuli.
Distinct characteristics of Tregs of newborns of healthy and allergic mothers.
Decreased allergy incidence in children supplemented with E. coli O83:K24:H31 and its possible modes of action.

Contact: 
jiri.hrdy@lf1.cuni.cz
LinkedIn