Studies of Human T Regulatory Cells

National Institute Of Allergy And Infectious Diseases

Investigators

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Abstract

Our primary interest is the development of novel mAbs to human Treg (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 FY23, we have focused our studies on a number of different areas: 1). Eight years ago, we established a CRADA with Boehringer-Ingelheim Pharmaceuticals to generate a panel of mAbs targeting expanded hTregs. It is widely documented that Treg play a deleterious role in the tumor microenvironment by suppressing anti-tumor effector T cells. Studies in murine models have suggested that deletion of Treg can result in an enhanced anti-tumor response. However, it has been difficult to identify a cell surface antigen that is uniquely expressed on Treg which can be targeted by a deleting monoclonal antibody. We immunized mice with human Treg cells which had been activated and expanded in vitro and screened the resulting hybridomas by flow cytometry for selective reactivity to activated Treg compared to similarly activated T effector cells. One hybridoma (2B010) which recognized CD25 was identified which had markedly increased reactivity to activated Treg. The affinity of 2B010 for CD25 was measured by surface plasmon resonance. The effects of 2B010 on IL-2-mediated STAT5 phosphorylation and on Treg suppressor function were evaluated. The reactivity of 2B010 antibody was also tested on T cells activated in vivo during xenogeneic graft versus host disease induced by injection of human PBMC into immunoincompetent NSG mice. The capacity of 2B010 to specifically delete Treg in the xeno-Graft Versus Host (GVHD) model and in the TME of CD34+ stem cell engrafted tumor bearing humanized mice was tested. 2B010 demonstrated enhanced reactivity only to Treg cells that had been expanded in culture for longer than 5 days, but displayed similar reactivity to a conventional anti-CD25 mAb on freshly expanded Treg suggesting that it might recognize a unique epitope on CD25 expressed only on activated Treg, but not expressed by activated Tconv. 2B010 displayed a relatively low affinity for CD25 compared to conventional anti-CD25 mAbs, did not block the binding of IL-2 in the STAT5 phosphorylation assay, and had no effect on the proliferation of Tconv or on Treg suppressor function. Most importantly, it selectively reacted with Treg activated in vivo during xeno-GVHD. Administration of 2B010 resulted in a selective deletion of Treg from mice undergoing xeno-GVHD. Administration of 2B010 to tumor bearing humanized mice also resulted in a profound depletion of Treg from the TME and activation of CD8+ T cells. Unfortunately, no effect on tumor growth was observed. Taken together, mAb 2B010 recognizes a unique epitope on CD25 expressed on activated Treg in vitro and in vivo. Administration of 2B010 results in selective depletion of Treg from an inflammatory site as well as the TME. 2B010 represents a useful candidate for treatment of patients with cancer either alone or together with check point inhibitors. 2). We have also addressed an important issue in human Treg biology. Foxp3 is regarded as the major transcription factor for T regulatory (Treg) cells and expression of Foxp3 is used to identify and quantitate Treg cells in mouse models. However, several studies have demonstrated that human CD4+ T conventional (Tconv) cells activated in vitro by T cell receptor (TCR) stimulation can express Foxp3. This observation has raised doubt as to the suitability of Foxp3 as a Treg marker in man. Helios, a member of the Ikaros gene family, has been shown to be expressed by 80-90% of human Foxp3+ Treg cells and can potentially serve as a marker of human Treg. Here, we confirm that Foxp3 expression is readily upregulated by Tconv upon TCR stimulation in vitro, while Helios expression is not altered. More importantly, we show that Foxp3 expression is not elevated by stimulation of hTconv in a humanized mouse model of graft versus host disease (GVHD) and in patients with a wide variety of acute and chronic inflammatory diseases including sickle cell disease, acute and chronic GVHD, systemic lupus erythematosus, as well as critical COVID-19. In all patients studied, an excellent correlation was observed between the percentage of CD4+ T cells expressing Foxp3 and the percentage expressing Helios. Taken together, these studies demonstrate that Foxp3 is not induced upon Tconv cell activation in vivo and that Foxp3 expression alone can be used to quantitate Treg cells in humans. Nevertheless, the combined use of Foxp3 and Helios expression provides a more reliable approach for the characterization of Treg in humans.