Studies of Human T Regulatory Cells

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

Our primary interest is the development of novel mAbs to hTreg with potential therapeutic utility and these studies are combined with studies on analysis of the unique biological properties of hTreg. Therapy with hTregs has already entered the clinic for treatment of GVHD, type I diabetes, and other diseases. It is therefore critical to characterize all aspects of hTreg phenotype and function. Therapeutic manipulation of hTreg function in vivo requires the development of novel biologics and mAbs as well as the appropriate in vivo models to test these reagents prior to administration to man. In Fy21, we have focused our studies on a number of different areas: 1). Six years ago, we established a CRADA with Boehringer-Ingelheim Pharmaceuticals to generate a panel of mAbs targeting expanded hTregs. Our initial strategy was to immunize mice with hTregs expanded in culture for 14 days, and then screen the resulting hybridomas both for binding to expanded hTregs, but not to activated Tconv cells. One large group of mAbs identified an epitope on CD25 that was expressed at high levels on expanded Treg, but at much lower levels on expanded Teff cells. These unique anti-CD25 mAbs did not inhibit T cell proliferation, modulate Treg suppression, or block IL-2 signaling (proliferation or STAT5 phosphorylation). Our efforts were focused on further characterization of the properties of two of the unique anti-CD25 mAbs (2B010 and 1C05) and their chimeric variants in vitro and in vivo. In vitro binding studies demonstrated that the chimeric mAbs and their Fc-silenced variants (LALA mutation) behaved identically to their parent mouse mAbs and demonstrated preferential binding to activated human T regulatory cells (CD4+CD25+Foxp3+helios+) compared to activated conventional T effector cells (CD4+CD25+Foxp3-helios-). Extensive studies were performed in vivo in two different humanized mouse models. In the PBMC reconstituted NSG model, both mouse and chimeric (2B010 and 1C05) depleted CD4+CD25+Foxp3+Helios+ T cells, only moderately depleted CD4+CD25+Foxp3-helios- T cells but did not deplete CD8+CD25+ T cells. The studies with the LALA mutation clearly demonstrated that the depletion was FcR dependent. In the human CD34+ reconstituted NOG-EXL model from Taconic, both mAbs and their humanized variants produced marked downmodulation of CD25 expression on all subsets. Some depletion of CD4+CD25+Foxp3+helios+ T cells was also observed, but the magnitude of the depletion was much less than that seen in the PBMC reconstituted NSG model. Our interpretation of these results is that the level of CD25 on both activated conventional cells and Treg in the CD34-NOG-EXL model is much less than that observed in the PBMC-NSG model due to the lack of GVHD in the former and is not sufficient to result in depletion. We elected to use the CD34-NOG-EXL model to test whether treatment with the mouse and chimeric mAbs will result in enhanced anti-tumor immunity as reflected in a decrease in tumor size or enhanced activation and cytokine production by CD4+Foxp3- or CD8+Foxp3- T cells. These experiments are now in progress. 2) We established a CRADA with related goals with Janssen Pharmaceuticals in Fy21. These studies are focused primarily on antigens that have been shown by us and other groups to be preferentially expressed on Treg such as LAYN, CD122, CCR4 and CCR8. Panels of mAbs are being generated by Janssen and will be screened to identify mAbs with selective or markedly increased preferential reactivity to human Tregs. The goal of these studies is to develop mAbs that can preferentially deplete Tregs in the tumor microenvironment. In addition, bi-specific mAbs are being developed that potentially can be utilized to increase the interactions of Treg with dendritic cells and thereby enhance their therapeutic potency. The goal of this aspect of the project is to develop drugs that can be used to enhance Treg function in autoimmune diseases. 3). We have also addressed an important issue in human Treg biology. One confounding factor in the study of human Tregs is that Tconv cells upregulate Foxp3 expression upon activation in vitro. However, it remains unclear whether this is the case in vivo. To investigate differences in phenotype between Tregs and activated Tconv cells, peripheral blood mononuclear cells (PBMCs) from healthy human donors were activated in vitro and in vivo and analyzed for expression of activation markers and cytokines using flow cytometry. For in vitro activation, PBMCs were stimulated in xenogeneic reactions with mouse splenocytes or with anti-CD3. For in vivo activation, human PBMC-engrafted NSG mice were used. During in vitro activation, there was an increase in Foxp3 expression by IL-2 producing, Helios- Tconv cells. In contrast, during the course of GVHD in vivo, activated Tconv cells, identified as CD25+ and Foxp3-, produced IL-2 but remained Foxp3- and Helios-. Foxp3+ Tregs did not produce IL-2 and remained predominantly Helios+. In conclusion, while expression of Foxp3 is not a reliable marker of activated human Tregs in vitro, it appears to be a specific marker of activated human Tregs in vivo as observed in this humanized mouse model. Using Foxp3 to distinguish human Tregs can improve diagnosis and treatment for autoimmune disorders and cancer.