Enhanced neuroinflammation is an important mechanism underlying perioperative neurocognitive disorders. Regulatory T cells (Tregs) play a crucial role in regulating systemic immune responses. The present study was aimed to investigate the participation of Tregs in the development of postoperative cognitive dysfunction (POCD).
Surgery-associated neurocognitive disorder was induced in 18-month-old mice subjected to internal fixation of tibial fracture. Morris water maze was used to examine mice cognitive function. Splenic Tregs were collected for RNA sequencing and flow cytometry. Levels of inflammatory factors in the circulation and hippocampus were measured by enzyme-linked immunosorbent assay. Protein presences of tight junction proteins were detected by immunofluorescence.
Surgery of internal fixation of tibial fracture induced cognitive impairment in aged mice, accompanied by elevated plasma levels of inflammatory factors and increased circulating Tregs. Transfusion of Tregs from young mice partially restored the structure of the blood-brain barrier and alleviated POCD in aged mice. Compared with young Tregs, differentially expressed genes in aged Tregs were enriched in tumor necrosis factor (TNF) signaling pathway and cytokine-cytokine receptor interaction. Flow cytometry revealed that aged Tregs had blunted functions under basal and stimulated conditions. Blockade of the CD25 epitope protected the blood-brain barrier structure, reduced TNF-α levels in the hippocampus, and improved surgery-associated cognition in aged mice.
Blocking peripheral regulatory T cells improves surgery-induced cognitive function in aged mice. Therefore, aged Tregs play an essential role in the occurrence of POCD.
© 2023. The Author(s).

Immune checkpoint inhibition (ICI) is a promising cancer therapy, which has progressed rapidly from a preclinical concept to clinical implementation. Commonly considered targets in ICI are CTLA-4, PD-1/PD-L1, and LAG-3, and the list grows. As ICI is generally only beneficial for a subset of patients, there is a need to select patients that are eligible for therapy as well as to monitor therapy response. There is growing interest to do this noninvasively, by molecular imaging with target-specific tracers. To this day, noninvasive imaging has focused on CTLA-4 and PD-1/PD-L1, while there is no noninvasive tool available to accurately assess LAG-3 expression in vivo. In this proof-of-concept study, we developed nanobodies, the smallest functional fragments from camelid heavy chain-only antibodies, to noninvasively evaluate mouse LAG-3 expression using single photon emission computed tomography (SPECT)/CT imaging. The in vitro characterization of 114 nanobodies led to the selection of nine nanobodies binding to mouse LAG-3. The injection of 99mTechnetium-labeled nanobodies in healthy mice showed specific uptake in immune peripheral organs like the spleen and lymph nodes, which was not observed in LAG-3 gene knock-out mice. Moreover, nanobody uptake could be visualized using SPECT/CT and correlated to the presence of LAG-3 as assessed in flow cytometry and immunohistochemistry. SPECT/CT scans of tumor bearing mice further confirmed the diagnostic potential of the nanobodies. These findings substantiate the approach to use nanobodies as a tool to image inhibitory immune checkpoints in the tumor environment.

The humanized immunocytokine, hu14.18-IL2 (ICp), leads to the immune cell-mediated destruction of GD2-expressing tumors in mouse models, resulting in potent antitumor effects with negligible IL2-related toxicity. In contrast, when ICp is used clinically, antitumor activity is accompanied by dose-limiting IL2-related toxicities. These species-specific differences in ICp toxicity may be linked to differential binding to mouse vs. human IL2 receptors (IL2Rs). We evaluated immunocytokines genetically engineered to preferentially bind either high-affinity αβγ-IL2Rs or intermediate-affinity βγ-IL2Rs. These ICs have the IL2 fused to the C-terminus of the IgG light chains rather than the heavy chains. We found that IC35, containing intact huIL2, maintained activation of human and mouse αβγ-IL2Rs but exhibited a 20-fold reduction in the ability to stimulate human βγ-IL2Rs, with no activation of mouse βγ-IL2Rs at the concentrations tested. The reduced ability of IC35 to stimulate human βγ-IL2Rs (associated with IL2-toxicities) makes it a potential candidate for clinical trials where higher clinical IC doses might enable better tumor targeting and increased antitumor effects with less toxicity. Contrastingly, ICSK (IC with an IL2 mutein that has enhanced binding to the IL2R β-chain) showed increased activation over ICp on mouse βγ-IL2Rs, with a dose-response curve similar to that seen with IC35 on human βγ-IL2Rs. Our data suggest that ICSK might be used in mouse models to simulate the anticipated effects of IC35 in clinical testing. Understanding the differences in species-dependent IL2R activation should facilitate the design of reagents and mouse models that better simulate the potential activity of IL2-based immunotherapy in patients.

Murine chronic graft-versus-host-disease (cGvHD) induced by injection of parental lymphocytes into F1 hybrids results in a disease similar to systemic lupus erythematosus. Here, we have used DBA/2 T cell injection into (C57BL/6 × DBA/2)F1 (BDF1) mice as a model system to test the prophylactic and therapeutic effects of interleukin-2 (IL-2)/anti-IL-2 immune complexes on the course of cGvHD. Our findings demonstrate that pretreatment with Treg inducing JES6/IL-2 complexes render BDF1 mice largely resistant to induction of cGvHD, whereas pretreatment with CD8+ T cell/NK cell inducing S4B6/IL-2 complexes results in a more severe cGvHD. In contrast, treatment with JES6/IL-2 complexes 4 weeks after induction had no beneficial effect on disease symptoms. However, similar treatment with S4B6/IL-2 complexes led to a significant amelioration of the disease. This therapeutic effect seems to be mediated by donor CD8+ T cells. The fact that a much stronger cGvHD is induced in BDF1 mice depleted of donor CD8+ T cells strongly supports this conclusion. The contrasting effects of the two different IL-2 complexes are likely due to different mechanisms.

Cancer dormancy is a clinical state in which residual tumor cells persist for long periods of time but do not cause detectable disease. In the mouse B cell lymphoma model (BCL1), dormancy can be induced and maintained by immunizing mice with a soluble form of the IgM expressed on the surface of the tumor cells. Immunization induces an anti-idiotype antibody response that maintains dormancy. Mice with dormant tumor have low numbers of BCL1 cells in their spleens that divide and are killed at the same rate. When the anti-Id antibodies wane, the tumor cells grow rapidly and kill the host. Spleens from tumor-bearing mice contain both effector (CD4+ and CD8+) and regulatory T cells (Tregs). In other tumor models, it has been reported that Tregs promote tumor progression by preventing effector cells from killing the tumor. In this report, we demonstrate that the tumor site with rapidly dividing BCL1 cells has fewer Tregs than the tumor site harboring dormant BCL1 cells. In both cases, the Tregs were equally suppressive in vitro. In spleens from mice with actively growing tumor, CD8+ but not CD4+ T cells were virtually absent. In vitro analysis demonstrated a tumor-mediated elimination of CD8+ T cells that was contact dependent and involved the caspase-3 pathway. Most importantly, we found that the BCL1 cells expressed characteristics of B10 regulatory B cells, i.e., they were CD1dhiCD5+ and secreted high levels of IL-10. These BCL1 tumor cells can inhibit anti-tumor immune responses by depleting CD8+ effector T cells.