Efficiency of Spermatogonial Dedifferentiation during Aging

Adult stem cells are critical for tissue homeostasis; therefore, the mechanisms utilized to maintain an adequate stem cell pool are important for the survival of an individual. In Drosophila, one mechanism utilized to replace lost germline stem cells (GSCs) is dedifferentiation of early progenitor cells. However, the average number of male GSCs decreases with age, suggesting that stem cell replacement may become compromised in older flies.Using a temperature sensitive allelic combination of Stat92E to control dedifferentiation, we found that germline dedifferentiation is remarkably efficient in older males; somatic cells are also effectively replaced. Surprisingly, although the number of somatic cyst cells also declines with age, the proliferation rate of early somatic cells, including cyst stem cells (CySCs) increases.These data indicate that defects in spermatogonial dedifferentiation are not likely to contribute significantly to an aging-related decline in GSCs. In addition, our findings highlight differences in the ways GSCs and CySCs age. Strategies to initiate or enhance the ability of endogenous, differentiating progenitor cells to replace lost stem cells could provide a powerful and novel strategy for maintaining tissue homeostasis and an alternative to tissue replacement therapy in older individuals

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

Characterization of germline stem cell behavior during aging in Drosophila melanogaster

Adult stem cells replenish tissues during normal cellular turnover or injury. A stem cell divides asymmetrically to produce one stem cell (a process called self-renewal), and one daughter cell that initiates differentiation. The mechanisms ensuring the balance of cells are critical for tissue homeostasis. Misregulation of stem cells during disease or aging can lead to impaired tissue maintenance and repair resulting in diminished life quality or death of an individual. Stem cells are located in a microenvironment, or niche, within a tissue. The niche, composed of other cells, ECM, and/or a basement membrane, provides extrinsic cues to stem cells that regulate self- renewal, maintenance, and survival. Therefore, analyses of stem cells should include the niche, to ensure that an observation is physiologically relevant. While in vivo studies of stem cells are critical, complex vertebrate tissues are difficult for 1) identifying stem cells 2) determining niche components 3) deciphering signaling pathways within stem cells, and between stem cells and the niche. Therefore, studies of stem cells within the context of disease or aging would add more hurdles to the research process. This dissertation presents research using the powerful model organism, Drosophila melanogaster, to study the effects of aging on germline stem cell (GSC) behavior in the testis, a well-characterized tissue. Chapter 2 shows that aging results in reduced GSC function due to extrinsic changes to the niche and intrinsic changes to the GSCs. Janus kinase -Signal Transducer and Activator of Transcription, Jak-STAT, signaling that regulates stem cell maintenance, was reduced during aging, and restoration of signaling led to a rescue of GSC number within the niche in aged animals. Male flies are able to replace lost GSCs through reversion (dedifferentiation) of early germline progenitor cells. Chapter 3 shows that GSC numbers are lower during aging possibly due to compromised reversion. Analysis of early cyst cells, one of the components of the niche, shows defects in response to reversion in aged flies. This dissertation summarizes that interactions between the stem cells and their niche dictate tissue health; changes to the niche and stem cells lead to decreased tissue homeostasis during agin

Characterization of germline stem cell behavior during aging in Drosophila melanogaster

Adult stem cells replenish tissues during normal cellular turnover or injury. A stem cell divides asymmetrically to produce one stem cell (a process called self-renewal), and one daughter cell that initiates differentiation. The mechanisms ensuring the balance of cells are critical for tissue homeostasis. Misregulation of stem cells during disease or aging can lead to impaired tissue maintenance and repair resulting in diminished life quality or death of an individual. Stem cells are located in a microenvironment, or niche, within a tissue. The niche, composed of other cells, ECM, and/or a basement membrane, provides extrinsic cues to stem cells that regulate self- renewal, maintenance, and survival. Therefore, analyses of stem cells should include the niche, to ensure that an observation is physiologically relevant. While in vivo studies of stem cells are critical, complex vertebrate tissues are difficult for 1) identifying stem cells 2) determining niche components 3) deciphering signaling pathways within stem cells, and between stem cells and the niche. Therefore, studies of stem cells within the context of disease or aging would add more hurdles to the research process. This dissertation presents research using the powerful model organism, Drosophila melanogaster, to study the effects of aging on germline stem cell (GSC) behavior in the testis, a well-characterized tissue. Chapter 2 shows that aging results in reduced GSC function due to extrinsic changes to the niche and intrinsic changes to the GSCs. Janus kinase -Signal Transducer and Activator of Transcription, Jak-STAT, signaling that regulates stem cell maintenance, was reduced during aging, and restoration of signaling led to a rescue of GSC number within the niche in aged animals. Male flies are able to replace lost GSCs through reversion (dedifferentiation) of early germline progenitor cells. Chapter 3 shows that GSC numbers are lower during aging possibly due to compromised reversion. Analysis of early cyst cells, one of the components of the niche, shows defects in response to reversion in aged flies. This dissertation summarizes that interactions between the stem cells and their niche dictate tissue health; changes to the niche and stem cells lead to decreased tissue homeostasis during agin

Increased proliferation of early cyst cells during aging.

<p>(A–D″) Immunofluorescence images of testes from young (A-A″; C-C″) and aged (B-B″; D-D″) flies stained for the hub (Fas3; red, outline), early cyst cells (Zfh-1; red) and labeled with EdU (green). Note EdU+, Zfh-1⁺ cells (arrowheads). Two merged 1 µm z-slices to represent majority of Zfh-1⁺ cells. (E) Graph of the percentage of Zfh-1⁺ cells in S-phase labeled by EdU in young and aged flies. Bracket with * shows statistically significant changes p<0.001. Scale bar: 10 µm. Genotype: <i>Stat92E^F/⁺</i> and <i>Stat92E^ts</i> (<i>Stat92E^F/Stat92E⁰⁶³⁴⁶</i>).</p

Stat92E localization in young flies during the reversion paradigm.

<p>(A–F′) Immunofluorescence images of testes from young OregonR, <i>Stat92E^F</i>, and <i>Stat92E^ts</i> flies stained for hub cells (Fas3, red, outline), early cyst cells (Traffic Jam [TJ], red), and Stat92E (green). (A–C′) at 18°C (D–F′) at 29°C for 2-days. Arrowheads point to Stat92E⁺TJ⁺ cells. Scale bars: 10 µm. Genotype: (A-A′,D-D′) <i>OregonR</i>; (B-B′,E-E′) <i>Stat92E^F/</i>+; (C-C′) <i>Stat92E^F/Stat92E⁰⁶³⁴⁶</i>; (F-F′) <i>Stat92E^F/Stat92E^J6C8</i>.</p

Decline in early cyst cells during aging.

<p>(A) Graph of the average number of Zfh-1⁺ cells in young and aged flies maintained at 18°C. Error bars represent standard error of the mean (SE). Genotypes: <i>Stat92E^F</i>/+ , <i>Stat92E^F/Stat92E⁰⁶³⁴⁶</i> and OregonR.</p

Average number of Zfh-1⁺ cells in testes from young and aged males and during dedifferentiation.

*<p>-Standard Error of the Mean.</p

Behavior of early cyst cells during dedifferentiation.

<p>(A) Graph of the average number of Zfh-1⁺ cells throughout the reversion paradigm in young and aged, <i>Stat92E^ts</i> flies. Error bars represent standard error of the mean (SE). Bracket with * shows statistically significant changes p<0.001. Genotype: <i>Stat92E^ts</i> (<i>Stat92E^F/Stat92E⁰⁶³⁴⁶</i>). (B–D′) Immunofluorescence images of testes from young flies throughout the reversion paradigm stained for the hub (Fas3; red, outline), late cyst cells (Eyes Absent [EyA]; red, arrowhead), and early cyst cells (Zfh-1; green). (B-B′) at 18°C, (C-C′) at 29°C for 2 days, and (D-D′) recovery at 18°C for another 2 days. Number of merged 1 µm z-slices to represent majority of Zfh-1⁺ cells for (A-A′) z = 4 (B-B′) z = 1 (C-C′) z = 2. Note changes in density of Zfh-1⁺ cells. Scale bars: 10 µm. Genotype: <i>Stat92E^F/Stat92E⁰⁶³⁴⁶</i>.</p

The effect of aging on germ line dedifferentiation in the <i>Drosophila</i> testis.

<p>(A) Schematic of early spermatogenesis and the <i>Stat92E^ts</i> reversion paradigm. GSCs (light green) and cyst stem cells (CySCs, light gray) surround the hub (red). A pair of CySCs envelopes each GSC, while cyst cells (dark gray) envelope gonialblasts and spermatogonial cysts (dark green). Red dots and branched structures represent a germ cell-specific organelle called the fusome. Jak-STAT signaling is required for stem cell maintenance. In flies carrying the temperature sensitive allele of <i>Stat92E</i>, GSCs differentiate at the restrictive temperature (29°C). Upon recovery at the permissive temperature (18°C), spermatogonia revert to GSCs. (B–G′) Immunofluorescence images of testes stained for Fasciclin3 (Fas3) (hub; red), alpha-spectrin (fusomes; red), and Vasa (germ cells; green) during dedifferentiation. (B-B′,E-E′) Spectrosomes (arrowheads) within GSCs and branched fusomes (arrows) within spermatogonia at 18°C in young (B-B′) or aged (E-E′) flies. (C-C′,F-F′) Branched fusomes within spermatogonia next to the hub (arrows) in flies shifted to 29°C for 2 days in young (C-C′) or aged (F-F′) flies. (D-D′) Spectrosomes within revertant GSCs (arrowheads) adjacent to the hub in young flies shifted back to 18°C for 5 days. (G-G′) Spermatogonia that contact the hub contain branched fusomes in testes from aged flies shifted back to 18°C for 5 days. (H) Quantification of testes containing at least one spectrosome in young and aged flies at 18°C (Permissive), 29°C (Restrictive), and recovery at 18°C (Restrictive-Permissive). (I) Average number of GSCs in testes that contained GSCs in young and aged flies raised at 18°C and after the recovery at 18°C. Error bars represent standard error of the mean (SE). Bracket with * shows statistically significant changes, p<0.001 Scale bar: 10 µm. Genotype: <i>Stat92E^F/Stat92E⁰⁶³⁴⁶</i>.</p