Bystander activation of memory T cells occurs via cytokine signaling alone in the absence of T cell receptor (TCR) signaling and provides a means of amplifying T cell effector responses in an antigen-nonspecific manner. While the role of Programmed Cell Death Protein 1 (PD-1) on antigen-specific T cell responses is extensively characterized, its role in bystander T cell responses is less clear. We examined the role of the PD-1 pathway during human and mouse non–antigen-specific memory T cell bystander activation and observed that PD-1 ⁺ T cells demonstrated less activation and proliferation than activated PD-1 ^– populations in vitro. Higher activation and proliferative responses were also observed in the PD-1 ^– memory population in both mice and patients with cancer receiving high-dose IL-2, mirroring the in vitro phenotypes. This inhibitory effect of PD-1 could be reversed by PD-1 blockade in vivo or observed using memory T cells from PD-1 ^–/– mice. Interestingly, increased activation through abrogation of PD-1 signaling in bystander-activated T cells also resulted in increased apoptosis due to activation-induced cell death (AICD) and eventual T cell loss in vivo. These results demonstrate that the PD-1/PD-Ligand 1 (PD-L1) pathway inhibited bystander-activated memory T cell responses but also protected cells from AICD.

Immune checkpoint inhibition (ICI) has emerged as one of the most powerful tools to reverse cancer induced immune suppression. Monoclonal antibodies (mAbs) targeting programmed cell death 1/programmed cell death ligand 1(PD-1/PD-L1) are FDA-approved and their clinical use is rapidly expanding. As opposed to the clinical paradigm, which can result in significant responses and toxicities, it has been difficult to reproduce these effects preclinically using mouse models. In large part, this is due to models, which employ rapidly growing ex vivo cultured transplantable tumor cell lines engrafted into young naïve inbred laboratory mice. However, another issue concerns the use and repeated application of xenogeneic reagents in mice (i.e., rat or hamster mAbs directed against mouse antigens at variance with clinical use of human or humanized mAbs). Building on our previous studies demonstrating that repeated administration of commonly used xenogeneic anti-PD-1 mAbs derived from both rat and hamster can induce fatal hypersensitivity in some tumor-bearing mice, we sought to compare these result with the effects of a mouse anti-mouse PD-1 mAb. Application of a murine anti-mouse PD-1 (clone: MuDX400) did not result in lethal anaphylaxis in the 4T1 tumor model. It also displayed superior antitumor effects in this and other tumor models, as it did not induce neutralizing antibody responses against the anti-PD-1 mAb, such as were observed when using xenogeneic anti-PD1 mAbs. These results demonstrate that more accurate preclinical modeling necessitates the use of mouse reagents mirroring the clinical scenario to ascertain long-term effects or toxicities, while avoiding xenogeneic responses, which do not occur clinically. Furthermore, these studies suggest a direct mechanism, whereby preclinical murine studies have often failed to recapitulate the clinical efficacy and toxicity of single agent checkpoint inhibition.

Background/Objectives: Novel mRNA vaccines have been successfully utilized to curtail the SARS-CoV-2 pandemic. However, the immunology underlying CoV2 vaccinations, particularly with repeated boosting, has not been properly characterized due to limitations in the preclinical modeling of SARS-CoV-2 infection/vaccinations as well as constantly changing vaccine formulations. The immunoregulatory aspects involved in such vaccine approaches remain unclear. Antibodies, due to inherent immunogenicity by VDJ gene rearrangement, have the potential to induce antibodies directed towards them called anti-idiotype antibodies, which can play a downregulatory role in responses. The paratope of some of these anti-idiotype antibodies can also act as a mirror to the original antigen, which, in the case of SARS-CoV-2 vaccines, would be to the spike protein and, therefore, also be capable of binding its target, ACE2, potentially causing adverse effects. Methods: To investigate if sequential SARS-CoV-2 mRNA vaccination can induce anti-idiotype antibody responses, K18 hACE2 transgenic mice were serially vaccinated with a SARS-CoV-2 mRNA construct to determine the kinetics of anti-spike and anti-ACE2 responses via custom-made ELISAs. Results: While sequential vaccination produced robust anti-spike responses, anti-ACE2 levels were also detected and gradually amplified with each boost. These anti-ACE2 antibodies persisted for 3 months after the final vaccination and showed evidence of hACE2 binding, as levels were lower in K18 mice in comparison to the wild type. Conclusions: These data would suggest that sequential SARS-CoV-2 mRNA vaccination has the potential to induce anti-ACE2 antibodies in mice, with each boost amplifying the amount of antibody.