31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

AGING-INDUCED INCREASED SUSCEPTIBILITY TO AUTOIMMUNITY IS DUE TO COMPROMISED NEGATIVE SELECTION IN THE THYMUS RATHER THAN DEFECTS IN REGULATORY T CELLS

Purpose: Immunotolerance generates protection against autoimmunity by deleting self reactive T cells in the thymus through negative selection as well as the generation of natural regulatory T cells (nTregs) that will help suppress autoimmunity in the periphery. Natural aging is associated with a progressive loss of FoxN1 and thymic atrophy, and is also believed to be associated with increased autoimmunity and an increase in suppressive FoxP3+ regulatory T cells (Tregs). Deletion of self-reactive T cells requires self-antigen presentation by medullary thymic epithelial cells (mTECs). We set out to determine if thymic aging, characterized by the progressive loss of FoxN1 and mTEC disruption, alters immunotolerance by influencing negative selection or impacting the generation of suppressive Treg cells. Methods: For thymus population experiments, we induce thymic atrophy in our FoxN1 conditional knockout mice using tamoxifen injections. We then determine expression of CD4+, CD8+, and CD4+8+ double positive, and CD25+FoxP3+ regulatory thymocytes using flow cytometry. The peripheral Treg data is assessed by adoptive transfer of total spleenocytes from young and aged wild type mice into young Rag2-/- mice. Cell populations are determined using flow cytometry. Results: We found that the loss of FoxN1 induced thymic atrophy is associated with an impairment of negative selection, where CD4+ and CD8+ are increased in the thymus while CD4+CD8+ double positive thymocytes are decreased. This indicates that the age atrophied thymus is not able to delete additional single positive thymocytes, and these may be self-reactive thymocytes. Additionally, we found that nTregs in the aged and atrophied thymus are increased in proportion and their suppressor function remains intact. Furthermore, we found that when we transfer aged spleenocytes, in which there are increased Treg and decreased pro-apoptotic Bim protein, and young spleenocytes separately into young Rag2 knockout mice, that the young periphery is able to restore both Bim levels and Treg levels to that of the young mice. Conclusions: We conclude that loss of FoxN1 disrupts thymic mTEC structure and impairs negative selection, which may lead to an increase in self-reactive T cells. However, thymic atrophy does not compromise peripheral Tregs. The function and number of peripheral Tregs is dependent on the micro-environment in which they stay

Thymic involution perturbs negative selection and leads to chronic inflammation

The ubiquitous presence of chronic low-level pro-inflammatory factors in elderly individuals (termed inflammaging) is a significant risk factor for morbidity and mortality. The etiology of inflammaging is largely unknown. Recent evidence has identified the persistent activation of immune cells, thought to arise from latent viral infections, as key contributors towards the development of a chronic inflammatory environment. However, the contribution of autoreactive T cells towards the development of inflammaging has yet to be investigated. Another pervasive feature of the aging process is the age-related involution of the thymus gland, which has been linked with a predisposition toward developing autoimmunity. In the present study, we determined how age-related thymic involution leads to the persistent release and activation of autoreactive T cells capable of inducing inflammaging. We utilized a FoxN1 conditional knock-out (FoxN1-cKO) mouse model that mimics thymic involution while maintaining a young periphery and naturally aged C57Bl/6 mice. We found that thymic involution leads to T cell activation shortly after thymic egress, which is accompanied by cellular infiltration into non-lymphoid tissues, elevated serum IL-6, and enhanced production of TNFα. Additionally, activated autoreactive T cell clones were detected in the periphery of FoxN1-cKO mice. We determined that a failure of negative selection, facilitated by decreased AIRE expression rather than impaired regulatory T cell (Treg) generation, and led to autoreactive T cell activation in the periphery. Furthermore, we have demonstrated that the young environment can reverse the age-related accumulation of Tregs but not inflammatory infiltration. Together, these findings identify thymic involution and the persistent activation of autoreactive T cells as a source of chronic age-related inflammation (inflammaging)

The age-related loss of FoxN1 and associated thymic involution leads to increased susceptibility to autoimmunity by impairing negative selection rather than influencing the generation of Tregs. (HEM4P.235)

Abstract The thymus maintains immunotolerance to self-antigen by deleting self-reactive T cells and generating nTregs. The thymus involutes with age, which is driven by the loss of FoxN1. Thymic involution is thought to be linked with increased susceptibility to autoimmune disease. However, how the age-related loss of FoxN1 induced thymic involution impacts autoimmunity remains unclear. We used a FoxN1 cKO mouse model that mimics natural thymic involution. We found impaired negative selection in the FoxN1 cKO thymus, evidenced by an increase in the frequency of CD4 and CD8 SP thymocytes and a decrease in Aire+ mTECs. Recent thymic emigrants from the FoxN1 cKO thymus have increased proliferation and are more often CD44+, indicating that they may be self-reactive T cells. Additionally, we found that nTreg frequency was increased in the thymus, but not in the spleen. We adoptively transferred aged wild-type splenocytes, in which there are a higher proportion of Treg cells into young Rag2-/- mice. We found that the young periphery was able to reverse Treg accumulation. Additionally, the adoptive transfer led to an increase in salivary infiltration independent of peripheral age. We conclude that loss of FoxN1 impairs negative selection, which may lead to an escape of self-reactive T cells. However, the age-related accumulation of Tregs depends on the age of the microenvironment in which they stay.</jats:p

THE AGE-RELATED LOSS OF FOXN1 AND SUBSEQUENT THYMIC INVOLUTION CONTRIBUTES TO AGE-RELATED AUTOIMMUNITY BY ALTERING IMMUNOTOLERANCE

The thymus is the organ responsible for developing a type of white blood cell called T cells. However, with age comes an increased susceptibility to T cell derived autoimmune disease. Additionally, the thymus progressively shrinks with increased age due to the progressive loss of the gene FoxN1. We want to determine if the loss of FoxN1leads to the thymus being unable to either delete or suppress autoimmune T cells. We utilize a mouse model that has a progressive loss of the FoxN1 gene. Our findings are significant because knowledge gained about the role of the FoxN1 gene with age-related autoimmune disease may lead to novel evidence-based gene therapy that targets the FoxN1 gene to help treat a wide range of autoimmune diseases associated with aging. Purpose (a): The thymus protects against autoimmune disease by generating immunotolernace to self-tissues. This is accomplished through the process of negative selection where self-reactive T cell clones are deleted and also by the generation of natural regulatory T cells (nTregs), which help suppress autoimmunity in the periphery. However, natural aging is associated with thymic atrophy driven by the progressive loss of the gene FoxN1. We wanted to determine if thymic aging impairs immunotolerance, either by disrupting negative selection or altering the generation of suppressive nTreg cells. Methods (b): We answered this question by utilizing a FoxN1 conditional knockout (FoxN1 cKO) mouse model that mimics natural thymic aging through the progressive loss of FoxN1. Results (c): We found that the loss of FoxN1 is associated with the impairment of negative selection characterized by increased single positive T cells and a decrease in Aire+ medullary thymic epithelial cells. Recent thymic emigrants from the FoxN1 cKO thymus have increased proliferation and are more often CD44+, indicating that they are antigen experienced and may be self-reactive T cells. Furthermore, we found that the frequency of nTregs was increased in the FoxN1 cKO thymus, but was normal in the spleen. Additionally, nTregs from the FoxN1 cKO thymus retained normal suppressive function. We adoptively transferred aged wild-type splenocytes, in which there are a higher proportion of Treg cells, into young Rag2-/- mice. We found that the young periphery was able to reverse the accumulation of Tregs. Additionally, the adoptive transfer led to an increase in infiltrating lymphocytes to the salivary gland, which was independent of peripheral age. Conclusions (d): We conclude that the loss of FoxN1 impairs negative selection, which may lead to an escape of self-reactive T cells. However, rather than being cell-intrinsic, the age-related accumulation of Tregs depends on the age of the peripheral microenvironment. These results indicate that the increased susceptibility to autoimmune disease observed with aging is likely due to defects in negative selection rather than changes in nTregs

Insights on Foxn1 Biological Significance and Usages of the &#8220;Nude&#8221; Mouse in Studies of T-Lymphopoiesis

<p>Mutation in the &#8220;nude&#8221; gene, i.e. the <i>FoxN1</i> gene, induces a hairless phenotype and a rudimentary thymus gland in mice (nude mouse) and humans (T-cell related primary immunodeficiency). Conventional <i>FoxN1</i> gene knockout and transgenic mouse models have been generated for studies of <i>FoxN1</i> 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 <i>loxP</i>-Cre(ER^T) 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 <i>FoxN1</i> in the thymus and utilizing nude and &#8220;second (conditional) nude&#8221; mouse models for studies of T-cell development and function. We also raise questions and propose further consideration of <i>FoxN1</i> functions and utilizing this mouse model for immune function studies.</p

Age-related thymic involution perturbs negative selection leading to autoreactive T Cells that induce chronic inflammation (inflammaging) (BA11P.141)

Abstract The presence of chronic low-level pro-inflammatory factors in elderly individuals (termed inflammaging) is a significant risk factor for morbidity and mortality. Recently, inflammaging has been partially attributed to the persistent activation of immune cells thought to arise from latent viral infection, but the contribution of activated autoreactive T cells towards the development of inflammaging remains unclear. To determine how age-related thymic involution leads to the persistent release and activation of autoreactive T cells capable of inducing inflammaging, we used a FoxN1 conditional knock-out (FoxN1-cKO) mouse model that mimics natural 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 cellular infiltration into non-lymphoid tissues and elevated IL-6 and TNFα levels. 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 but not inflammatory infiltration. Together, these findings identify thymic involution and the persistent activation of autoreactive T cells as a source of chronic age-related inflammation (inflammaging).</jats:p

Aging of thymic epithelial progenitor pool is determined by the p63-FoxN1 regulatory axis (HEM4P.233)

Abstract The postnatal thymic epithelial progenitor (TEP) pool is proposed to be regulated by the p63 and FoxN1 genes through proliferation and differentiation, respectively. However, the combined role of these two genes in the aging TEP is still a mystery. Evidence from murine models has elucidated contrasting roles of the p63 isoforms during the aging process. We found that TAp63+, but not ΔNp63+, thymic epithelial cells (TECs) were increased with age, accompanied with increased senescence associated β-gal clusters and p21+ TECs. Senescent clusters also developed after intrathymic infusion of exogenous TAp63 cDNA into young wild-type mice. Using our conditional FoxN1 gene knockout mouse model to disrupt TEP differentiation accelerated this senescent phenotype to early middle age. However, upon infusion of exogenous FoxN1 cDNA into aged wild-type mice resulted in only an increase in ΔNp63+ TECs, but no change in TAp63+ TECs in the partially rejuvenated aged thymus. Interestingly, using a novel FoxN1 transgenic mouse model to enhance TEP differentiation, ΔNp63+ TECs were decreased in young thymus. Additionally, the TAp63+ population contained a high percentage of phosphorylated-p53 and apoptotic TECs, but showed no changes in BrdU-labeled proliferation. As a result, FoxN1 controlled TEC differentiation as a bottleneck to determine TEP pool via affecting TAp63 and ΔNp63 levels. Thus, TEC homeostasis during aging has been determined through the p63-FoxN1 regulatory axis.</jats:p

Age-related Thymic Involution Perturbs Negative Selection Leading to Autoreactive T Cells That Induce Chronic Inflammation (Inflammaging)

Research Appreciation Day Award Winner - 2015 Graduate School of Biomedical Sciences Awards - 1st Place Oral PresentationResearch Appreciation Day Award Winner - 2015 Department of Cell Biology and Immunology - 1st Place Oral PresentationThe presence of chronic low-level pro-inflammatory factors in elderly individuals (termed inflammaging) is a significant risk factor for morbidity and mortality. Recently, inflammaging has been partially attributed to the persistent activation of immune cells thought to arise from latent viral infection, but the contribution of activated autoreactive T cells towards the development of inflammaging remains unclear. To address our hypothesis that age-related thymic involution leads to the persistent release and activation of autoreactive T cells capable of inducing inflammaging, we performed experiments including: adoptive transfer, kidney capsule transplantation, and tetramer detection of autoreactive T cells on a FoxN1 conditional knock-out (FoxN1-cKO) mouse model that mimics natural 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 and elevated serum IL-6 and TNFα levels. 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 but not inflammatory infiltration. Together, these findings identify thymic involution and the persistent activation of autoreactive T cells as a source of chronic age-related inflammation (inflammaging)