Regulatory T Cells in Humanized Immune System Mice

Regulatory T cells (Tregs) are a critical component of the immune system for regulating inflammatory responses. In the context of immune-mediated diseases, such as cancer or graft-versus-host disease (GvHD), Tregs can either drive or suppress disease pathogenesis. This two-sided role for Tregs and their impact on such a broad spectrum of diseases makes them an important focus of study in immuno-oncology and autoimmunity.

Preclinical models of these diseases ideally replicate this crucial component of the immune system so that the interaction between pro-inflammatory and anti-inflammatory immune cells can be evaluated. In order to best capture the impact of the human immune system on disease in preclinical studies, it is therefore important to select a model where all the components of the immune system that may be impacted by your study agent are present. In the context of cancer, this will often include Tregs. Human Tregs are reconstituted in Taconic Biosciences' huNOG-EXL mice, which are NOG mice that additionally express human GM-CSF and human IL-3 and are engrafted with CD34+ hematopoietic stem cell (HSC). Case studies describing the study of Tregs in the context of cancer will be discussed in this article.

Regulatory T Cell Markers and Function

Tregs are a subset of CD4+ T cells that makes up approximately 1-10% of human peripheral blood mononuclear cells in healthy individuals. Common markers used to identify human Tregs are CD3 and CD4 (to isolate T cells) and FoxP3+, CD25+, CD127- to refine the Treg population.

FoxP3 is a key transcription factor for Treg function. It directly regulates the expression of between 700-1,400 genes and, indirectly, about 10x more by complexing with binding partners. CD25 is the high-affinity IL-2Rα chain. In Tregs, IL-2 signaling activates the JAK3/STAT5 pathway that turns on transcription of the FOXP3 locus. Expression of CD25 correlates with FoxP3 levels and suppressive activity of Tregs¹. CD127, the IL-7 receptor, is a negative marker of Tregs, while it is expressed on effector and memory T cells. Its expression is inversely correlated with both CD25 and FoxP3 expression. Indeed, FoxP3 interacts with the CD127 promoter and is hypothesized to repress activation of this locus².

Tregs are Found in Blood, Spleen, and Bone Marrow of Humanized Immune System Mice

Taconic's huNOG-EXL mice reconstitute many human immune cell types following engraftment with HSCs. This includes T and B cells as well as myeloid cells (but very few or no NK cells). Within the T cell compartment, many studies have looked at the presence and frequency of Tregs under both healthy and diseased conditions in immune-rich organs such as the peripheral blood, spleen, and bone marrow.

Healthy Mice

A 2020 publication from Roche engrafted Taconic's NOG-EXL animals with CD34+ HSC in-house, then measured the frequency of various immune cell subpopulations in the bone marrow and spleen of these mice at 16 weeks post-engraftment. In this study, Tregs were identified as CD3+ CD4+ CD8- CD25+ FoxP3+ cells within the hCD45+ compartment. Tregs were approximately 1% of the hCD4+ T cell population in both bone marrow and spleen; CD4+ T cells comprised about 60% of the human T cells in both organs. In comparison, humanized NOG mice (which do not express hIL-3 or hGM-CSF) were also studied and almost no Tregs were detected in either the bone marrow or spleen of these mice at the same timepoint. This frequency of Tregs was accompanied by an elevated serum level of the Treg-recruiting chemokine CCL22 in the stem cell-engrafted NOG-EXL over NOG animals³.

Another case study used cyTOF to characterize the immune profile of the blood of humanized NOG-EXL animals at a later timepoint: 21 weeks post-engraftment. In this analysis, Tregs were characterized as CD4+ CD25+ CD127- T cells. Effector Tregs were the most prevalent subset of Tregs observed, over naïve and terminal effector Tregs, at ~6.2% of hCD45+ cells. In total, Tregs comprised ~6.7% of all hCD45+ cells.

Recently, Taconic partnered with Certis Oncology to further characterize the immune profile of huNOG-EXL animals. Taconic engrafted HSC from multiple donors into cohorts of 5 mice each, and Certis evaluated the presence of Tregs in the peripheral blood at 17-18 weeks post-engraftment. The average frequency of CD25+ CD127lo FoxP3+ Treg of the CD4+ T cell population ranged from 10.3% to 16.1%; the range of CD4+ T cells ranged from 60.7% to 76.2%.

Despite the expected variability that can be observed between the studies described in this Insight and within huNOG-EXL mice overall, Tregs have been repeatedly observed in this model and huNOG-EXL is therefore a suitable model for studying Tregs.

Tumor-bearing Mice

Tregs are also sustained in tumor-bearing mice at 23 weeks post-engraftment (n=3 donors, n=12 mice/donor). In a Taconic internal study, Tregs were identified as 6.90 - 11.18% of the hCD45+ compartment in the peripheral blood, using the markers CD4+ FoxP3+ to identify this subpopulation⁴. These Tregs are also able to infiltrate the tumor microenvironment (TME), where Tregs comprised an average of approximately 20% of T cells in the TME.

Tregs Respond Variably to Pharmacologic and Immunologic Interventions in the Context of Cancer

Other studies have evaluated intratumoral Tregs and their modulation in response to therapeutic intervention. While Tregs are consistently observed in huNOG-EXL animals, their response to therapeutic intervention varies. Several publications have looked at modulation of the Treg compartment in the TME of lung and breast cancers.

A study published by researchers from Tesaro and AnaptysBio evaluated Tregs in the context of PD-1 or LAG-3 blockade in the A549 non-small cell lung cancer (NSCLC) model engrafted into huNOG-EXL. Ten days following tumor inoculation, Tregs (hCD45+ hCD3+ hCD4+ hFoxP3+) were 3% of the total hCD45+ cell population in the blood. While the frequency and absolute Treg cell count were not reported, the relative frequency of intratumoral Tregs was not impacted by treatment⁵.

A subsequent paper from Tesaro used the MDA-MB-436 human breast cancer cell line to study the impact of the PARP inhibitor niraparib on the TME. A previous study looking at PARP inhibition in a murine model of ovarian cancer noted an increase of both effector and regulatory T cells in the tumor following treatment; however, in the current study, any increase in intratumoral CD3+ FoxP3+ Tregs in the human breast cancer cell-derived xenograft (CDX) following treatment was not statistically significant⁶.

In conclusion, huNOG-EXL is a suitable preclinical model for studying Tregs, especially in the context of cancer. The frequency of Tregs in the peripheral blood of huNOG-EXL mice is similar to that observed in human peripheral blood, and these cells can traffic into the TME of CDX and patient-derived xenograft (PDX).

References:

1. Lu, L.; Barbi, J.; Pan, F. The Regulation of Immune Tolerance by FOXP3. Nat Rev Immunol 2017, 17 (11), 703-717.

2. Liu, W.; Putnam, A. L.; Xu-yu, Z.; Szot, G. L.; Lee, M. R.; Zhu, S.; Gottlieb, P. A.; Kapranov, P.; Gingeras, T. R.; Groth, B. F. de St.; Clayberger, C.; Soper, D. M.; Ziegler, S. F.; Bluestone, J. A. CD127 Expression Inversely Correlates with FoxP3 and Suppressive Function of Human CD4+ T Reg Cells. J Exp Medicine 2006, 203 (7), 1701-1711.

3. Maser, I.-P.; Hoves, S.; Bayer, C.; Heidkamp, G.; Nimmerjahn, F.; Eckmann, J.; Ries, C. H. The Tumor Milieu Promotes Functional Human Tumor-Resident Plasmacytoid Dendritic Cells in Humanized Mouse Models. Front Immunol 2020, 11, 2082.

4. Richardson, J. R.; MacBride, M.; Andino, E.; Wilwol, M.; Bethmann, S.; Knapp, T.; Shako, E.; Volden, P. Abstract 2935: Defining Variation within a next Gen Humanized Immune System Mouse Model of Human Lung Cancer in the Context of a Checkpoint Inhibitor Efficacy Study. Tumor Biol 2021, 2935-2935.

5. Ghosh, S.; Sharma, G.; Travers, J.; Kumar, S.; Choi, J.; Jun, H. T.; Kehry, M.; Ramaswamy, S.; Jenkins, D. TSR-033, a Novel Therapeutic Antibody Targeting LAG-3 Enhances T Cell Function and the Activity of PD-1 Blockade in Vitro and in Vivo. Mol Cancer Ther 2018, 18 (3), molcanther.0836.2018.

6. Wang, Z.; Sun, K.; Xiao, Y.; Feng, B.; Mikule, K.; Ma, X.; Feng, N.; Vellano, C. P.; Federico, L.; Marszalek, J. R.; Mills, G. B.; Hanke, J.; Ramaswamy, S.; Wang, J. Niraparib Activates Interferon Signaling and Potentiates Anti-PD-1 Antibody Efficacy in Tumor Models. Sci Rep-uk 2019, 9 (1), 1853.