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 Fy22, we have focused our studies on a number of different areas: 1). Seven 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 have been focused on further characterization of the properties of one of the unique anti-CD25 mAbs (2B010) in vitro and in vivo. Extensive studies were performed in vivo in two different humanized mouse models. In the PBMC reconstituted NSG model, in which the mice develop xeno-GVHD, 2B010 reacted to a much greater extent with CD4+CD25+Foxp3+ T cells compared to activated CD4+CD25+Foxp3- T cells. 2B010 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 depletion was FcR dependent. One possible explanation for the selective depletion of Treg from NSG mice reconstituted with PBMC is that the Tregs in the transferred PBMC population down-modulate the expression of CD25 on the CD4+Foxp3- effector cells thereby facilitating engraftment of the human T cells. To address this possibility, we transferred purified CD4+Foxp3- T cells alone of with purified Treg into NSG mice and treated both groups with 2B010 one week later. When CD4+Foxp3- cells were transferred alone, marked upregulation of CD25 expression was observed. Treatment with 2B010 resulted in a lower level of CD25 expression, but a non-significant decrease in the percentages of CD4+Foxp3- T cells. Even though the levels of CD25 expression were decreased by treatment with 2B010, the CD4+Foxp3- T cells appeared to be highly activated as the levels of PD-1 and CTLA-4 were similar to animals treated with the isotype control mAb. A markedly different effect of 2B010 on T cell activation was observed in animals that received both CD4+Foxp3- T cells and Treg. Significant depletion of CD4+CD25+Foxp3+ cells was observed in 2B010 treated mice. Co-transfer of Treg resulted in marked suppression of the activation of the CD4+Foxp3- effectors. The suppression of activation by Treg was significantly reversed in 2B010 treated mice consistent with a functional depletion of Treg. In the human CD34+ reconstituted NOG-EXL model where xeno-GVHD is not observed, 2B010 produced marked downmodulation of CD25 expression on all subsets. 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. We elected to use the CD34-NOG-EXL model to test whether treatment with 2B010 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. CD34+ stem cell reconstituted NSG mice were implanted orthotopically with MDA MB231 breast cancer cells. A significant number of human CD45+ cells were detected in the TIL and CD4+ T cells were dominant in the control animals while CD8+ T cells predominated in the 2B010 treated mice. The majority of the CD4+ T cells in the TIL of control mice were Foxp3+Helios+ Treg and very few CD4+CD25+Foxp3- could be detected. Treatment with 2B010 resulted in a highly significant depletion of Treg and marked enhancement of granzyme B production by CD8+ T cells. Unfortunately, no reduction on tumor size was observed. 2). We have also addressed an important issue in human Treg biology. Foxp3 is regarded as the major transcription factor for 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 TCR stimulation can express Foxp3. This observation has raised considerable doubt as to the suitability of Foxp3 as a bona fide 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. We confirm that Foxp3 expression is readily upregulated by Treg upon TCR stimulation in vitro, while Helios expression is not altered. More importantly, we show that Foxp3 expression is not elevated in a wide variety of acute and chronic inflammatory diseases including, sickle cell disease, acute and chronic graft versus host disease, systemic lupus erythematosus, as well as severe 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 fail-safe approach for the characterization of Treg in humans. 3) We established a CRADA with Janssen Pharmaceuticals in FY21 and continued these studies in FY22. 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 have been generated by Janssen and have been screened by us. We have identified mAbs with selective or markedly increased 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.