Thymic involution and the subsequent amplified release of autoreactive T cells increase the susceptibility toward developing autoimmunity, but whether they induce chronic inflammation with advanced age remains unclear. The presence of chronic low-level pro-inflammatory factors in elderly individuals (termed inflammaging) is a significant risk factor for morbidity and mortality in virtually every chronic age-related disease. To determine how thymic involution leads to the persistent release and activation of autoreactive T cells capable of inducing inflammaging, we used a FoxN1 conditional knockout (FoxN1-cKO) mouse model that induces accelerated thymic involution while maintaining a young periphery. We found that thymic involution leads to T cell activation shortly after thymic egress, which is accompanied by a chronic inflammatory phenotype consisting of cellular infiltration into non-lymphoid tissues, increased TNFα production and elevated serum IL-6. Autoreactive T cell clones were detected in the periphery of FoxN1-cKO mice. A failure of negative selection, facilitated by decreased expression of Aire rather than impaired regulatory T cell (Treg) generation, led to autoreactive T cell generation. Furthermore, the young environment can reverse age-related Treg accumulation in naturally aged mice, but not inflammatory infiltration. Together, these findings identify thymic involution and the persistent activation of autoreactive T cells as a contributing source of chronic inflammation (inflammaging).

CCR8 is a chemokine receptor expressed principally on regulatory T cells (Treg) and is known to be critical for CCR8 Treg-mediated immunosuppression. Recent studies have demonstrated that CCR8 is uniquely upregulated in human tumor-resident Tregs of patients with breast, colon, and lung cancer when compared with normal tissue-resident Tregs. Therefore, CCR8 tumor-resident Tregs are rational targets for cancer immunotherapy. Here, we demonstrate that mAb therapy targeting CCR8 significantly suppresses tumor growth and improves long-term survival in colorectal tumor mouse models. This antitumor activity correlated with increased tumor-specific T cells, enhanced infiltration of CD4 and CD8 T cells, and a significant decrease in the frequency of tumor-resident CD4CCR8 Tregs. Tumor-specific CD8 T cells displayed lower expression of exhaustion markers as well as increased functionality upon restimulation. Treatment with anti-CCR8 mAb prevented induction and suppressive function of Tregs without affecting CD8 T cells. Initial studies explored a combinatorial regimen using anti-CCR8 mAb therapy and a -based immunotherapy. Anti-CCR8 mAb therapy synergized with-based immunotherapy to significantly delay growth of established tumors and to prolong survival. Collectively, these findings identify CCR8 as a promising new target for tumor immunotherapy and provide a strong rationale for further development of this approach, either as a monotherapy or in combination with other immunotherapies. Inhibition of CCR8 represents a promising new cancer immunotherapy strategy that modulates tumor-resident regulatory T cells to enhance antitumor immunity and prolong patient survival. .

Mutation in the “nude” gene, i.e. the FoxN1 gene, induces a hairless phenotype and a rudimentary thymus gland in mice (nude mouse) and humans (T-cell related primary immunodeficiency). Conventional FoxN1 gene knockout and transgenic mouse models have been generated for studies of FoxN1 gene function related to skin and immune diseases, and for cancer models. It appeared that FoxN1's role was fully understood and the nude mouse model was fully utilized. However, in recent years, with the development of inducible gene knockout/knockin mouse models with the loxP-Cre(ERT) and diphtheria toxin receptor-induced cell abolished systems, it appears that the complete repertoire of FoxN1's roles and deep-going usage of nude mouse model in immune function studies have just begun. Here we summarize the research progress made by several recent works studying the role of FoxN1 in the thymus and utilizing nude and “second (conditional) nude” mouse models for studies of T-cell development and function. We also raise questions and propose further consideration of FoxN1 functions and utilizing this mouse model for immune function studies.

The interaction between T cells and the central nervous system (CNS) in homeostasis and injury has been recognized being both pathogenic (CD4+ T-helper 1 - Th1, Th17 and γδT) and ameliorative (Th2 and regulatory T cells - Tregs). However, in-depth studies aimed to elucidate the precise in the aged microenvironment and the dichotomous role of Tregs have just begun and many aspects remain unclear. This is due, not only to a mutual dependency and reciprocal causation of alterations and diseases between the nervous and T cell immune systems, but also to an inconsistent aging of the two systems, which dynamically changes with CNS injury/recovery and/or aging process. Cellular immune system aging, particularly immunosenescence and T cell aging initiated by thymic involution - sources of chronic inflammation in the elderly (termed inflammaging), potentially induces an acceleration of brain aging and memory loss. In turn, aging of the brain via neuro-endocrine-immune network drives total body systemic aging, including that of the immune system. Therefore, immunotherapeutics including vaccination and “protective autoimmunity” provide promising means to rejuvenate neuro-inflammatory disorders and repair CNS acute injury and chronic neuro-degeneration. We review the current understanding and recent discoveries linking the aging immune system with CNS injury and neuro-degeneration. Additionally, we discuss potential recovery and rejuvenation strategies, focusing on targeting the aging T cell immune system in an effort to alleviate acute brain injury and chronic neuro-degeneration during aging, via the “thymus-inflammaging-neurodegeneration axis”.

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FoxN1 is cell-autonomously expressed in skin and thymic epithelial cells (TECs), essential for their development. Inborn mutation of FoxN1 results in hair follicle and TEC development failure, whereas insufficient postnatal FoxN1 expression induces thymic atrophy, resulting in declined T lymphopoiesis. Although upregulating FoxN1 expression in the aged FoxN1-declined thymus rejuvenates T lymphopoiesis, whether its over- and ectopic-expression in early life is beneficial for T lymphopoiesis is unknown. Using our newly generated Rosa26-STOPflox–FoxN1 mice, in which over- and ectopic-expression of FoxN1 can be induced by various promoter-driven Cre-mediated deletions of the roadblock STOPflox in early life, we found that K14Cre-mediated inborn FoxN1 overexpression induced neonatal lethality, exhibited abnormal permeability in the skin and abnormal nursing. Ubiquitous deletion of the STOPflox mediated by progressive uCreERT leakage in juvenile mice affected thymus and bone marrow normality, resulting in an increased ratio of medullary/cortical TECs, along with declined T and B lymphopoiesis. Although the K5CreERT-mediated FoxN1 overexpression mice had a normal lifespan, induction of K5CreERT activation in juveniles adversely influenced total thymoycte development and produced ichthyosis-like skin. Therefore, FoxN1 has temporal and tissue-specific activity. Over- and ectopic-expression of FoxN1 in early life adversely influence immature TEC, T and B cell, and skin epithelial development.

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Introduction: Virtually all tumors contain somatic mutations that can result in novel antigenic sequences that may be targeted by the host cellular immune response. Some of these mutations occur in preferential regions of specific genes commonly referred to as hotspot mutations. Hotspot mutations are commonly shared by cancer patients both within and across multiple tumor types. These hotspot mutations often confer loss or gain of function contributing to oncogenesis, which makes them promising therapeutic targets. One such mutation commonly found in several human tumor types is an aspartic acid substitution for glycine at position 12 (G12D) in KRAS. This same mutation occurs in the CT26 murine colorectal tumor model. To determine if expression of the KRAS G12D sequence in a bacterial immunotherapy vector can control tumor growth in the CT26 murine model, the Advaxis Listeria monocytogenes (Lm)-based platform was engineered to express a 21-amino acid KRAS sequence peptide containing the G12D mutation (Lm-Hot KRAS_G12D). In addition, we evaluated control of tumor growth using an ADXS-HOT construct (ADXS-503) that expresses multiple shared human hotspot and tumor-associated antigens, including the G12D KRAS. The ADXS-HOT clinical program is comprised of several Lm-based immunotherapies designed to target multiple shared hotspot and tumor-associated antigens commonly found in specific cancer types. In this study, we demonstrate control of tumor growth in a mouse model by targeting a commonly shared hotspot mutation using an Lm-based immunotherapy. Results: We show that the Lm-HOT KRAS_G12D therapy significantly delayed tumor growth and improved long-term survival in the murine CT26 colon carcinoma model. This response was associated with an increase in the frequency of tumor infiltrating antigen-specific CD8 T cells and γδ T cells within the tumor microenvironment and a decrease in the frequency of intratumoral regulatory T cells (Tregs). Furthermore, tumor-specific CD8 T cells displayed lower expression of exhaustion markers as well as increased functionality upon restimulation. Interestingly, our proprietary ADXS-503 (a clinical ADXS-HOT construct) which includes KRAS G12D as one of its multiple targets, was also capable of significantly suppressing tumor growth in the CT26 tumor model. Conclusion: These results suggest that our ADXS-HOT platform is a promising approach to target shared hotspot mutations. That ADXS Lm constructs targeting a single hotspot mutation can significantly control tumor growth whether it is in a single or multi-target construct. These data describe an exciting translatable discovery with the potential for broad utility across multiple tumor types and patients who share common hotspot mutations. Citation Format: Daniel Villarreal, Brandon Coder, Susan Armington, Andrew L'Huillier, Cristina Mottershead, Elena Filippova, Nithya Thambi, Kim Ramos, David Balli, Robert Petit, Michael Princiotta. Targeting shared hotspot cancer mutations with a Listeria monocytogenes immunotherapy induce potent anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-149.