CD4+ Foxp3+ Tregs are an independent cell lineage and their developmental progression during thymic development depends on IL-2R signaling. However, the role of IL-2R signaling during thymic Treg development remains only partially understood. The current study assessed the contribution of IL-2 in the expansion and functional programming of developing Tregs. In the absence of IL-2Rβ signaling, predominately CD4+ CD25- Foxp3lo T cells were found and these cells exhibited somewhat lower expression of the proliferative marker Ki67. These immature Tregs, which represent products of failed development, were also found in normal mice and characterized by markedly lower expression of several Treg functional molecules. IL-2R signaling, therefore, is required for the progression, functional programming, and expansion of Tregs during thymic development. An IL-2R signaling mutant that lowers STAT5 activation readily supported Treg functional programming but Treg proliferation remained somewhat impaired. The requirement for IL-2 during thymic Treg expansion was best illustrated in mixed chimeras where the Tregs with mutant IL-2Rs were forced to compete with WT Tregs during their development. Tregs with impaired IL-2R signaling were more prevalent in the thymus than spleen in these competitive experiments. The general effectiveness of mutant IL-2Rs to support thymic Treg development is partially accounted for by a heighten capacity of thymic Tregs to respond to IL-2. Overall our data support a model where limiting IL-2R signaling is amplified by thymic Tregs to readily support their development and functional programming whereas these same conditions are not sufficient to support peripheral Treg homeostasis.

Recent work suggests that IL-2 and IL-15 induce distinctive levels of signaling through common receptor subunits and that such varied signaling directs the fate of antigen-activated CD8+ T cells. Here we directly examined proximal signaling by IL-2 and IL-15 and CD8+ T cell primary and memory responses as a consequence of varied CD122-dependent signaling. Initially, IL-2 and IL-15 induced similar pStat5 and pS6 activation, but these activities were only sustained by IL-2. Transient IL-15-dependent signaling is due to limited expression of IL-15Rα. To investigate the outcome of varied CD122 signaling for CD8+ T cell responses in vivo, OT-I T cells were utilized from mouse models where CD122 signals were attenuated by mutations within the cytoplasmic tail of CD122 or intrinsic survival function was provided in the absence of CD122 expression by transgenic Bcl-2. In the absence of CD122 signaling, generally normal primary response occurred, but the primed CD8+ T cells were not maintained. In marked contrast, weak CD122 signaling supported development and survival of TCM but not TEM cells. Transgenic expression of Bcl-2 in CD122−/− CD8+ T cells also supported the survival and persistence of TCM cells but did not rescue TEM development. These data indicate that weak CD122 signals readily support TCM development largely through providing survival signals. However, stronger signals, independent of Bcl-2, are required for TEM development. Our findings are consistent with a model whereby low, intermediate, and high CD122 signaling support TCM memory survival, TEM programming, and terminal TEFF differentiation, respectively.

Resting central Tregs (cTregs) and activated effector Tregs (eTregs) are required for self-tolerance, but the heterogeneity and relationships within and between phenotypically distinct subsets of cTregs and eTregs are poorly understood. By extensive immune profiling and deep sequencing of TCRβ V-regions, two subsets of cTregs, based on expression of Ly-6C, and three subsets of eTregs, based on distinctive expression of CD62L, CD69, and CD103, were identified. Ly-6C+ cTregs exhibited lower basal activation, expressed on average lower affinity TCRs, and less efficiently developed into eTregs when compared to Ly-6C− cTregs. The dominant TCR Vβs of Ly-6C+ cTregs were shared by eTregs at a low frequency. A single TCR clonotype was also identified that was largely restricted to Ly-6C+ cTregs, even under conditions that promoted the development of eTregs. Collectively, these findings indicate that some Ly-6C+ cTregs may persist as a lymphoid-specific subset, with minimal potential to develop into highly activated eTregs, while other cTregs readily develop into eTregs. In contrast, subsets of CD62Llo eTregs showed higher clonal expansion and were more highly inter-related than cTreg subsets based on their TCRβ repertoires, but exhibited varied immune profiles. The CD62Llo CD69− CD103− eTreg subset displayed properties of a transitional intermediate between cTregs and more activated eTreg subsets. Thus, eTregs subsets appear to exhibit substantial flexibility, likely in response to environmental cues, to adopt defined immune profiles that are expected to optimize suppression of autoreactive T cells.

Natural killer (NK) cells are a promising cellular therapy for cancer, with challenges in the field including persistence, functional activity, and tumor recognition. Briefly, priming blood NK cells with recombinant human (rh)IL-12, rhIL-15, and rhIL-18 (12/15/18) results in memory-like NK cell differentiation and enhanced responses against cancer. However, the lack of available, scalable Good Manufacturing Process (GMP)–grade reagents required to advance this approach beyond early-phase clinical trials is limiting. To address this challenge, we developed a novel platform centered upon an inert tissue factor scaffold for production of heteromeric fusion protein complexes (HFPC). The first use of this platform combined IL-12, IL-15, and IL-18 receptor engagement (HCW9201), and the second adds CD16 engagement (HCW9207). This unique HFPC expression platform was scalable with equivalent protein quality characteristics in small- and GMP-scale production. HCW9201 and HCW9207 stimulated activation and proliferation signals in NK cells, but HCW9207 had decreased IL-18 receptor signaling. RNA sequencing and multidimensional mass cytometry revealed parallels between HCW9201 and 12/15/18. HCW9201 stimulation improved NK cell metabolic fitness and resulted in the DNA methylation remodeling characteristic of memory-like differentiation. HCW9201 and 12/15/18 primed similar increases in short-term and memory-like NK cell cytotoxicity and IFNγ production against leukemia targets, as well as equivalent control of leukemia in NSG mice. Thus, HFPCs represent a protein engineering approach that solves many problems associated with multisignal receptor engagement on immune cells, and HCW9201-primed NK cells can be advanced as an ideal approach for clinical GMP-grade memory-like NK cell production for cancer therapy.

A network of mechanisms operates to maintain tolerance in the gut mucosa. Although CD103 marks many lymphoid cells within the gut, its direct functional role in intestinal tolerance is poorly understood. CD103 may be part of a redundant pathway as CD103−/− mice do not exhibit autoimmunity. To reduce such redundancy, CD103−/− mice were crossed to mice (designated Y3) whose T cells expressed a mutant IL-2Rβ chain that lowers IL-2R signaling. Unlike overtly healthy Y3 mice, all Y3/CD103−/− mice rapidly developed severe colitis. The large intestine of these mice contained an increase in CD4+ Th1 and Th17 effector cells and a reduced ratio of regulatory T cells (Tregs). Importantly, colitis was effectively prevented by the transfer of wild type (WT) Tregs into Y3/CD103−/− mice. Impaired intestinal tolerance was not attributed to an obvious lack of CD103-dependent gene regulation or intestinal homing/retention by Tregs nor a lack of functional activities typically associated with CD103+ dendritic cells (DCs), such as peripheral induced Treg (pTreg) development or imprinting CCR9 and α4β7 homing molecules on Treg and T effector cells. Transcriptome analysis of Tregs was consistent with altered homeostasis due to impaired IL-2Rβ-dependent signaling with minimal dysregulation added by the absence of CD103. Rather the absence of CD103 functioned to alter the localization of the cells within the gut microenvironment that may alter Treg homeostasis. Thus, IL-2Rβ-dependent signaling and CD103 normally cooperate through distinctive processes to promote Treg homeostasis and immune tolerance.