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Regulatory T cells (Tregs) are an immunosuppressive cell population that inhibits immune cell function to maintain peripheral tolerance and homeostasis. We have previously shown that Treg-- response to interferon gamma (IFNγ) is required for response to cancer immunotherapy. We aimed to broaden these findings and determine the impact of Treg response to IFNγ and the IFNγ-inducing cytokine IL12, during viral infection. Mice with Treg-restricted deletion of the IFNγ receptor (IFNGR1, Ifngr1L/LFoxp3Cre-YFP) or the IL12 receptor β2 subunit (IL12Rβ2, Il12b2L/LFoxp3Cre-YFP), were infected with the acute (Armstrong), or chronic (Clone 13), strain of lymphocytic choriomeningitis virus (LCMV). Surprisingly, Treg response to IFNγ but not IL12 induced helper T cell 1 (TH1)-like polarization (expression of T-bet, CXCR3 and IFNγ) of Tregs in mice with chronic LCMV infection. Importantly, this effector-like TH1 Treg state was required for adequate suppression of effector T cells during LCMV infection. Additionally, during LCMV infection, Treg-restricted deletion of IFNGR1 not only prevented TH1-like polarization but also reverted these Tregs to a TH2-like state (expression of GATA-3, CCR4 and IL-4). scRNAseq of viral antigen-specific CD8+ T cells from Clone 13 infected mice showed that Ifngr1-deficient Tregs favored CD8+ T cell memory. These findings were confirmed in an Armstrong/Listeria monocytogenes-GP33 memory model. Further, Ifngr1 deletion from Tregs enhanced response to CD8+ T cell-mediated vaccination when challenged with Clone 13. These findings provide fundamental insight on how Tregs sense inflammatory cues from the environment (IFNγ) during viral infection to prevent overt tissue damage and shape the memory response. Supported by grants from NIH (F32 CA247004-01, T32 CA082084, P01 AI108545, R35 CA263850, R01 CA203689, R01 DK130897, P30 DK120531, P30 CA047904, R01 HL107380, R01 CA206517, R01 AI138504)

We have shown that murine regulatory T cells (Tregs) produce the cytokine interferon gamma (IFNγ) during anti-PD1 immunotherapy and that IFNγ-driven Treg dysfunction is necessary for tumor clearance by anti-PD1. However, it is unknown how Treg production of IFNγ dictates response to cancer immunotherapy. It has been shown that IL12-induced production of IFNγ is required for response to anti-PD1 therapy. However, it has yet to be determined whether Tregs are a key responder to IL12. Thus, elucidating the interplay of IL12, IFNγ and Tregs in the tumor microenvironment (TME) is essential for understanding the mechanistic events required for response to immunotherapy. Our lab has generated two novel murine models that allow for Treg-restricted deletion of Ifng (IfngL/LFoxp3Cre-YFP) or Il12rb2 (Il12rb2L/LFoxp3Cre-YFP). These murine models were used to assess the contribution of Treg-derived IFNγ and response to IL12 on the growth of various syngeneic tumor models and response to checkpoint blockade, vaccination and tumor-specific antibodies. Surprisingly, mice with Ifng-deficient Tregs were completely resistant to anti-PD1 immunotherapy. Preliminary experiments suggest that IFNγ production by Tregs is an early requirement to produce an effector-like Treg state in order to mediate an effective anti-tumor response. Mice with Il12rb2-deficient Tregs had diminished therapeutic response to anti-PD1. These data suggest that IFNγ producing Tregs are required to shift the balance from an immunosuppressive TME to one that favors the reinvigoration of the anti-tumor response generated by cancer immunotherapies. Future studies will determine the mechanisms of resistance and whether IFNγ+ Tregs predict response to immunotherapy.

The inhibitory receptor Lymphocyte activation gene 3 (LAG3; CD223) plays a key role in regulating T cell activation and tolerance. LAG3 can be expressed on CD4+ FoxP3+ regulatory T cells (Tregs) and conventional effector T cells (Tcons). We found that LAG3 is highly expressed by CD4+ FoxP3+ Tregs in the CNS of mice with experimental autoimmune encephalitis, a mouse model of multiple sclerosis, leading us to examine LAG3 function on CD4+ FoxP3+ Treg in EAE. To study LAG3 function on Tregs in EAE, we crossed mice in which LAG3 is absent on the cell surface of CD4+ FoxP3+ Tregs with 2D2 T cell receptor (TCR) transgenic specific for myelin oligodendrocyte glycoprotein (MOG35-55). Surprisingly, 100% of the 2D2 TCR transgenic mice lacking LAG3 on Tregs developed spontaneous EAE. In marked contrast, none of the 2D2 control mice developed EAE. Similarly, non-TCR transgenic mice lacking LAG3 on Tregs developed more severe induced EAE (by immunization with MOG35-55 in CFA) compared to controls (max clinical score 3.5±0.3 in LAG3 deficient vs. 2.8±0.1 in control SEM). Histological analysis revealed significantly more lesions in both brain and spinal cord of LAG3 deficient mice (mean number in spinal cord 226.5 ± 10.57 LAG3 deficient vs. 51.4 ± 40.81 in controls SEM). Ex vivo analysis at the peak of disease showed that disease severity was accompanied by an increase in the pathogenicity of Tcons producing high levels of IFN-γ and granzyme B. Although no significant differences were observed in CNS infiltration of Tregs, LAG3 deficient Tregs produced IFN-γ and granzyme B, suggesting that they are no longer protective, but instead contribute to CNS disease. These findings point to a key role for LAG3 in controlling Treg function and maintenance of tolerance in the CNS.

The mechanism and stimulatory requirements of regulatory T cell (Treg)-mediated suppression are still unclear. To assess the requirement for Treg stimulation by cognate peptide:MHC, we used T cells from OTII and AND TCR transgenic mice that are specific for and restricted by distinct, noncrossreactive peptide:MHC combinations. This allowed us to independently activate Tregs and their conventional T cell (Tconv) targets. Surprisingly, we found that suppression can occur in the absence of peptide:MHC-mediated stimulation of Tregs. This suppression was Treg dependent and not due to cold target inhibition. Using Rag1−/− TCR transgenic T cells, we show that regulation of Tconv proliferation by heterogeneous Tregs is not due to alloreactivity or crossreactivity. Finally, using anti-TCR-Vβ8-coated microbeads and Vβ8− Tregs, we show that TCR stimulation-independent suppression can occur in the absence of APCs. These data suggest that Tregs may possess constitutive regulatory activity that can be mediated in the absence of cognate peptide:MHC-TCR stimulation.

Lymphocyte activation gene 3 (LAG-3) is a CD4-related, activation-induced cell surface molecule expressed by various lymphoid cell types and binds to MHC class II with high affinity. We have previously shown that LAG-3 negatively regulates the expansion of activated T cells and T cell homeostasis, and is required for maximal regulatory T cell function. In this study, we demonstrate for the first time that LAG-3 is also expressed on CD11clow/B220+/PDCA-1+ plasmacytoid dendritic cells (pDCs). Lag3 expression, as determined by real time PCR, was ∼10-fold greater in pDCs than in either regulatory T cells or activated T effector cells. Activated pDCs also generate ∼5 times more sLAG-3 than activated T cells. LAG-3-deficient pDCs proliferate and expand more than wild-type pDCs in vivo in response to the TLR9 ligand, CpG. However, the effect of LAG-3 appears to be selective as there was no effect of LAG-3 on the expression of MHC class II, TLR9, and chemokine receptors, or on cytokine production. Lastly, adoptive transfer of either Lag3+/+ or Lag3−/− T cells plus or minus Lag3+/+ or Lag3−/− pDCs defined a role for LAG-3 in controlling pDC homeostasis as well as highlighting the consequences of deregulated Lag3−/− pDCs on T cell homeostasis. This raised the possibility of homeostatic reciprocity between T cells and pDCs. Collectively, our data suggests that LAG-3 plays an important but selective cell intrinsic and cell extrinsic role in pDC biology, and may serve as a key functional marker for their study.

The CD3ε proline-rich sequence (PRS) binds to the cytosolic adaptor molecule Nck after TCR ligation. It has been proposed that this interaction is essential for immunological synapse formation and T cell activation. To assess the physiological importance of the CD3ε PRS, we have generated mice that lack this motif (CD3ε.PRSM). Pull-down experiments demonstrated the inability of Nck to bind to the CD3ε PRS in thymocytes from mutant mice after TCR ligation. Surprisingly, no differences were observed in the number and percentage of T cell subsets in the thymus and spleen, and there was no apparent defect in positive or negative selection. Furthermore, the proliferative response of CD3ε.PRSM T cells to staphylococcal enterotoxin B and anti-CD3 Ab was normal. TCR surface expression, constitutive internalization, and Ag-induced down-modulation were also normal. These data suggest that the interaction between the CD3ε PRS and Nck, or any other Src homology 3 domain-containing molecule, is not essential for T cell development and function.

Lymphocyte homeostasis is a central biological process that is tightly regulated. However, its molecular and cellular control is poorly understood. We show that aged mice deficient in lymphocyte activation gene 3 (LAG-3), an MHC class II binding CD4 homologue, have twice as many T cells as wild-type controls. CD4+ and CD8+ LAG-3-deficient T cells showed enhanced homeostatic expansion in lymphopenic hosts, which was abrogated by ectopic expression of wild-type LAG-3, but not by a signaling-defective mutant. In addition, in vivo treatment with anti-LAG-3 mAb resulted in enhanced T cell expansion to a level comparable to that in LAG-3-deficient cells. This deregulation of T cell homeostasis also resulted in the expansion of multiple cell types, including B cells, macrophages, granulocytes, and dendritic cells. Lastly, regulatory T cells were dependent on LAG-3 for their optimal control of T cell homeostasis. Our data suggest that LAG-3 negatively regulates T cell homeostasis by regulatory T cell-dependent and independent mechanisms.

Lymphocyte activation gene (LAG)-3 (CD223) is a CD4-related activation-induced cell surface molecule that binds to MHC class II molecules with high affinity and negatively regulates T cell expansion and homeostasis. In this study, we show that LAG-3 inhibits CD4-dependent, but not CD4-independent, T cell function via its cytoplasmic domain. Although high affinity interaction with MHC class II molecules is essential for LAG-3 function, tailless LAG-3 does not compete with CD4 for ligand binding. A single lysine residue (K468) within a conserved “KIEELE” motif is essential for interaction with downstream signaling molecules. These data provide insight into the mechanism of action of this important T cell regulatory molecule.

Inhibitory receptors control immune responses preventing exacerbated T cell activation and limiting antitumor immunity. LAG3 co-expression with PD1 phenotypically marks functionally exhausted tumor-specific T cells, however LAG3 expression and function is itself regulated by ADAM metalloproteinase-mediated cell surface cleavage. To investigate the impact of LAG3 shedding on T cells within tumors, a conditional knock-in mouse was generated that results in a non-cleavable form of LAG3 (LAG3.NC). LAG3.NC CD4Cre, LAG3.NC ThPOKCreERT2 and LAG3.NC E8ICre mice (restricting LAG3.NC to all T cells, CD4+ T cells and CD8+ T cells, respectively) exhibit enhanced LAG3 expression on the respective T cell subsets infiltrating MC38 tumors. Upon therapeutic administration of anti-PD1, MC38 tumor-bearing wild-type mice show significant tumor regression and 40% become tumor-free, resulting in long-term survival. LAG3.NC CD4Cre and LAG3.NC ThPOKCreERT2 resist anti-PD1 therapy and succumb to tumor growth. However, this phenotype is not observed in LAG3.NC E8ICre mice, resulting in a similar frequency of tumor-free mice to controls. CD8+ TIL isolated from LAG3.NC CD4Cre mice do not show enhanced IFN-g/TNF-a production or proliferation as observed in controls or LAG3.NC E8ICremice following anti-PD1. Failure to mediate an antitumor immune response by LAG3.NC CD4Cre, but not LAG3.NC E8ICre mice, suggests that LAG3 cleavage on CD4+ T cells, including Tregs, is necessary for CD8+ TIL to elicit an effective immune response with anti-PD1. Selective ADAM10 inhibition in vivo, preventing LAG3 shedding, also reduces cytokine release, proposing that metalloproteinase-mediated LAG3 cleavage limits the efficacy of the antitumor immune response.

We wish to retract the article titled “Human Regulatory T Cells Require IL-35 To Mediate Suppression and Infectious Tolerance” by Vandana Chaturvedi, Lauren W. Collison, Clifford S. Guy, Creg J. Workman, and Dario A. A. Vignali, The Journal of Immunology , 2011, 186: [6661–6666][1]. A recent