Amrutesh Puranik, Ph.D.

Abstract
Purpose: We previously showed that elevated frequencies of peripheral blood CD3+CD4+CD127-GARP-CD38+CD39+ T-cells was associated with checkpoint immunotherapy resistance in metastatic melanoma patients. In the present study we sought to further investigate this population of ectoenzyme expressing T-cells (Teee). Expeirmental Design: Teee derived from the peripheral blood of metastatic melanoma patients were evaluated by bulk RNA-seq and flow cytometry. The presence of Teee in the… Show more

Abstract
Purpose: We previously showed that elevated frequencies of peripheral blood CD3+CD4+CD127-GARP-CD38+CD39+ T-cells was associated with checkpoint immunotherapy resistance in metastatic melanoma patients. In the present study we sought to further investigate this population of ectoenzyme expressing T-cells (Teee). Expeirmental Design: Teee derived from the peripheral blood of metastatic melanoma patients were evaluated by bulk RNA-seq and flow cytometry. The presence of Teee in the tumor microenviroment was assessed using publically available single-cell RNA-seq datasets of melanoma, lung, and bladder cancer along with multispectral immunofluorescent imaging of melanoma patient FFPE specimens. Suppressive function of Teee was determined by an in vitro autologous suppression assay.

Results: Teee had phenotypes associated with proliferation, apoptosis, exhaustion, and high expression of inhibitory molecules. Cells with a Teee gene signature were present in melanoma, lung cancer and bladder cancer patients' tumors. CD4+ T-cells co-expressing CD38 and CD39 in the tumor microenvironment were preferentially associated with Ki67- CD8+ T-cells. Co-culture of patient Teee with autologous T-cells resulted in decreased proliferation of target T-cells. High baseline intra-tumoral frequencies of Teee were associated with checkpoint immunotherapy resistance and poor overall survival in metastatic melanoma patients.

Conclusions: These results demonstrate that a novel population of CD4+ T-cells co-expressing CD38 and CD39 is found both in the peripheral blood and tumor of melanoma patients and is associated with checkpoint immunotherapy resistance. Show less

Abstract
Background
Peripheral vascular diseases may induce chronic ischemia and cellular injury distal to the arterial obstruction. Cellular senescence involves proliferation arrest in response to stress, which can damage neighboring cells. Renal artery stenosis (RAS) induces stenotic-kidney dysfunction and injury, but whether these arise from cellular senescenceand their temporal pattern remain unknown.

Methods
Chronic renal ischemia was induced in transgenic INK-ATTAC and… Show more

Abstract
Background
Peripheral vascular diseases may induce chronic ischemia and cellular injury distal to the arterial obstruction. Cellular senescence involves proliferation arrest in response to stress, which can damage neighboring cells. Renal artery stenosis (RAS) induces stenotic-kidney dysfunction and injury, but whether these arise from cellular senescenceand their temporal pattern remain unknown.

Methods
Chronic renal ischemia was induced in transgenic INK-ATTAC and wild type C57BL/6 mice by unilateral RAS, and kidney function (in vivo micro-MRI) and tissue damage were assessed. Mouse healthy and stenotic kidneys were analyzed using unbiased single-cell RNA-sequencing. To demonstrate translational relevance, cellular senescence was studied in human stenotic kidneys.

Results
Using intraperitoneal AP20187 injections starting 1, 2, or 4 weeks after RAS, selective clearance of cells highly expressing p16Ink4a attenuated cellular senescence and improved stenotic-kidney function; however, starting treatment immediately after RAS induction was unsuccessful. Broader clearance of senescent cells, using the oral senolytic combination dasatinib and quercetin, in C57BL/6 RAS mice was more effective in clearing cells positive for p21 (Cdkn1a) and alleviating renal dysfunction and damage. Unbiased, single-cell RNA sequencing in freshly dissociated cells from healthy and stenotic mouse kidneys identified stenotic-kidney epithelial cells undergoing both mesenchymal transition and senescence. As in mice, injured human stenotic kidneys exhibited cellular senescence, suggesting this process is conserved.

Conclusions
Maladaptive tubular cell senescence, involving upregulated p16 (Cdkn2a), p19 (Cdkn2d), and p21 (Cdkn1a) expression, is associated with renal dysfunction and injury in chronic ischemia. These findings support development of senolytic strategies to delay chronic ischemic renal injury. Show less

Kidney-resident macrophages promote angiogenesis and prevent fibrosis in ischemic kidneys.

Renal artery stenosis (RAS) caused by narrowing of arteries is characterized by microvascular damage. Macrophages are implicated in repair and injury, but the specific populations responsible for these divergent roles have not been identified. Here, we characterized murine kidney F4/80+CD64+ macrophages in three transcriptionally unique populations. Using fate-mapping and parabiosis studies, we… Show more

Kidney-resident macrophages promote angiogenesis and prevent fibrosis in ischemic kidneys.

Renal artery stenosis (RAS) caused by narrowing of arteries is characterized by microvascular damage. Macrophages are implicated in repair and injury, but the specific populations responsible for these divergent roles have not been identified. Here, we characterized murine kidney F4/80+CD64+ macrophages in three transcriptionally unique populations. Using fate-mapping and parabiosis studies, we demonstrate that CD11b/cint are long-lived kidney-resident (KRM) while CD11chiMϕ, CD11cloMϕ are monocyte-derived macrophages. In a murine model of RAS, KRM self-renewed, while CD11chiMϕ and CD11cloMϕ increased significantly, which was associated with loss of peritubular capillaries. Replacing the native KRM with monocyte-derived KRM using liposomal clodronate and bone marrow transplantation followed by RAS, amplified loss of peritubular capillaries. To further elucidate the nature of interactions between KRM and peritubular endothelial cells, we performed RNA-sequencing on flow-sorted macrophages from Sham and RAS kidneys. KRM showed a prominent activation pattern in RAS with significant enrichment in reparative pathways, like angiogenesis and wound healing. In culture, KRM increased proliferation of renal peritubular endothelial cells implying direct pro-angiogenic properties. Human homologs of KRM identified as CD11bintCD11cintCD68+ increased in post-stenotic kidney biopsies from RAS patients compared to healthy human kidneys, and inversely correlated to kidney function. Thus, KRM may play protective roles in stenotic kidney injury through expansion and upregulation of pro-angiogenic pathways. Show less

Identified tissue resident macrophage progenitors

Visceral adiposity is a risk factor for cardiovascular disorders, type 2 diabetes mellitus (T2D) and associated metabolic diseases. Sub-cutaneous fat is believed to be intrinsically different from visceral fat. To understand molecular mechanisms involved in metabolic advantages of fat transplantation, we studied a rat model of diet-induced adiposity. Adipokine genes (Adiponectin, Leptin, Resistin and Visfatin) were expressed at 10,000 to a million-fold lower in visceral fat depot as compared to… Show more

Visceral adiposity is a risk factor for cardiovascular disorders, type 2 diabetes mellitus (T2D) and associated metabolic diseases. Sub-cutaneous fat is believed to be intrinsically different from visceral fat. To understand molecular mechanisms involved in metabolic advantages of fat transplantation, we studied a rat model of diet-induced adiposity. Adipokine genes (Adiponectin, Leptin, Resistin and Visfatin) were expressed at 10,000 to a million-fold lower in visceral fat depot as compared to peripheral (thigh/chest) fat depots. Interestingly, autologous transplantation of visceral fat to subcutaneous sites resulted in increased gene transcript abundance in the grafts by 3 weeks post-transplantation, indicating the impact of local (residence) factors influencing epigenetic memory. We show here that active transcriptional state of adipokine genes is linked with glucose mediated recruitment of enzymes that regulate histone methylation. Adipose depots have “residence memory” and autologous transplantation of visceral fat to sub-cutaneous sites offers metabolic advantage Show less

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