Life Sciences Research for Lifelong Health

Publications rahul-roychoudhuri

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Akt inhibition enhances expansion of potent tumor-specific lymphocytes with memory cell characteristics.
Crompton JG, Sukumar M, Roychoudhuri R, Clever D, Gros A, Eil RL, Tran E, Hanada K, Yu Z, Palmer DC, Kerkar SP, Michalek RD, Upham T, Leonardi A, Acquavella N, Wang E, Marincola FM, Gattinoni L, Muranski P, Sundrud MS, Klebanoff CA, Rosenberg SA, Fearon DT, Restifo NP

Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) results in complete regression of advanced cancer in some patients, but the efficacy of this potentially curative therapy may be limited by poor persistence of TIL after adoptive transfer. Pharmacologic inhibition of the serine/threonine kinase Akt has recently been shown to promote immunologic memory in virus-specific murine models, but whether this approach enhances features of memory (e.g., long-term persistence) in TIL that are characteristically exhausted and senescent is not established. Here, we show that pharmacologic inhibition of Akt enables expansion of TIL with the transcriptional, metabolic, and functional properties characteristic of memory T cells. Consequently, Akt inhibition results in enhanced persistence of TIL after adoptive transfer into an immunodeficient animal model and augments antitumor immunity of CD8 T cells in a mouse model of cell-based immunotherapy. Pharmacologic inhibition of Akt represents a novel immunometabolomic approach to enhance the persistence of antitumor T cells and improve the efficacy of cell-based immunotherapy for metastatic cancer.

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Cancer research, 75, 1538-7445, 296-305, 2015

PMID: 25432172

Type I cytokines synergize with oncogene inhibition to induce tumor growth arrest.
Acquavella N,Clever D,Yu Z,Roelke-Parker M,Palmer DC,Xi L,Pflicke H,Ji Y,Gros A,Hanada K,Goldlust IS,Mehta GU,Klebanoff CA,Crompton JG,Sukumar M,Morrow JJ,Franco Z,Gattinoni L,Liu H,Wang E,Marincola F,Stroncek DF,Lee CC,Raffeld M,Bosenberg MW,Roychoudhuri R,Restifo NP

Both targeted inhibition of oncogenic driver mutations and immune-based therapies show efficacy in treatment of patients with metastatic cancer, but responses can be either short lived or incompletely effective. Oncogene inhibition can augment the efficacy of immune-based therapy, but mechanisms by which these two interventions might cooperate are incompletely resolved. Using a novel transplantable BRAF(V600E)-mutant murine melanoma model (SB-3123), we explored potential mechanisms of synergy between the selective BRAF(V600E) inhibitor vemurafenib and adoptive cell transfer (ACT)-based immunotherapy. We found that vemurafenib cooperated with ACT to delay melanoma progression without significantly affecting tumor infiltration or effector function of endogenous or adoptively transferred CD8(+) T cells, as previously observed. Instead, we found that the T-cell cytokines IFNγ and TNFα synergized with vemurafenib to induce cell-cycle arrest of tumor cells in vitro. This combinatorial effect was recapitulated in human melanoma-derived cell lines and was restricted to cancers bearing a BRAF(V600E) mutation. Molecular profiling of treated SB-3123 indicated that the provision of vemurafenib promoted the sensitization of SB-3123 to the antiproliferative effects of T-cell effector cytokines. The unexpected finding that immune cytokines synergize with oncogene inhibitors to induce growth arrest has major implications for understanding cancer biology at the intersection of oncogenic and immune signaling and provides a basis for design of combinatorial therapeutic approaches for patients with metastatic cancer. Cancer Immunol Res; 3(1); 37-47. ©2014 AACR. See related commentary by Riddell, p. 23.

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Cancer immunology research, 3, 2326-6074, 37-47, 2015

PMID: 25358764

Identification of the genomic insertion site of Pmel-1 TCR α and β transgenes by next-generation sequencing.
Ji Y, Abrams N, Zhu W, Salinas E, Yu Z, Palmer DC, Jailwala P, Franco Z, Roychoudhuri R, Stahlberg E, Gattinoni L, Restifo NP

The pmel-1 T cell receptor transgenic mouse has been extensively employed as an ideal model system to study the mechanisms of tumor immunology, CD8+ T cell differentiation, autoimmunity and adoptive immunotherapy. The 'zygosity' of the transgene affects the transgene expression levels and may compromise optimal breeding scheme design. However, the integration sites for the pmel-1 mouse have remained uncharacterized. This is also true for many other commonly used transgenic mice created before the modern era of rapid and inexpensive next-generation sequencing. Here, we show that whole genome sequencing can be used to determine the exact pmel-1 genomic integration site, even with relatively 'shallow' (8X) coverage. The results were used to develop a validated polymerase chain reaction-based genotyping assay. For the first time, we provide a quick and convenient polymerase chain reaction method to determine the dosage of pmel-1 transgene for this freely and publically available mouse resource. We also demonstrate that next-generation sequencing provides a feasible approach for mapping foreign DNA integration sites, even when information of the original vector sequences is only partially known.

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PloS one, 9, 1932-6203, e96650, 2014

PMID: 24827921

Open Access

Inhibiting glycolytic metabolism enhances CD8+ T cell memory and antitumor function.
Sukumar M,Liu J,Ji Y,Subramanian M,Crompton JG,Yu Z,Roychoudhuri R,Palmer DC,Muranski P,Karoly ED,Mohney RP,Klebanoff CA,Lal A,Finkel T,Restifo NP,Gattinoni L

Naive CD8+ T cells rely upon oxidation of fatty acids as a primary source of energy. After antigen encounter, T cells shift to a glycolytic metabolism to sustain effector function. It is unclear, however, whether changes in glucose metabolism ultimately influence the ability of activated T cells to become long-lived memory cells. We used a fluorescent glucose analog, 2-NBDG, to quantify glucose uptake in activated CD8+ T cells. We found that cells exhibiting limited glucose incorporation had a molecular profile characteristic of memory precursor cells and an increased capacity to enter the memory pool compared with cells taking up high amounts of glucose. Accordingly, enforcing glycolytic metabolism by overexpressing the glycolytic enzyme phosphoglycerate mutase-1 severely impaired the ability of CD8+ T cells to form long-term memory. Conversely, activation of CD8+ T cells in the presence of an inhibitor of glycolysis, 2-deoxyglucose, enhanced the generation of memory cells and antitumor functionality. Our data indicate that augmenting glycolytic flux drives CD8+ T cells toward a terminally differentiated state, while its inhibition preserves the formation of long-lived memory CD8+ T cells. These results have important implications for improving the efficacy of T cell-based therapies against chronic infectious diseases and cancer.

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The Journal of clinical investigation, 123, 1558-8238, 4479-88, 2013

PMID: 24091329

Open Access

Retinoic acid controls the homeostasis of pre-cDC-derived splenic and intestinal dendritic cells.
Klebanoff CA,Spencer SP,Torabi-Parizi P,Grainger JR,Roychoudhuri R,Ji Y,Sukumar M,Muranski P,Scott CD,Hall JA,Ferreyra GA,Leonardi AJ,Borman ZA,Wang J,Palmer DC,Wilhelm C,Cai R,Sun J,Napoli JL,Danner RL,Gattinoni L,Belkaid Y,Restifo NP

Dendritic cells (DCs) comprise distinct populations with specialized immune-regulatory functions. However, the environmental factors that determine the differentiation of these subsets remain poorly defined. Here, we report that retinoic acid (RA), a vitamin A derivative, controls the homeostasis of pre-DC (precursor of DC)-derived splenic CD11b(+)CD8α(-)Esam(high) DCs and the developmentally related CD11b(+)CD103(+) subset within the gut. Whereas mice deprived of RA signaling significantly lost both of these populations, neither pre-DC-derived CD11b(-)CD8α(+) and CD11b(-)CD103(+) nor monocyte-derived CD11b(+)CD8α(-)Esam(low) or CD11b(+)CD103(-) DC populations were deficient. In fate-tracking experiments, transfer of pre-DCs into RA-supplemented hosts resulted in near complete conversion of these cells into the CD11b(+)CD8α(-) subset, whereas transfer into vitamin A-deficient (VAD) hosts caused diversion to the CD11b(-)CD8α(+) lineage. As vitamin A is an essential nutrient, we evaluated retinoid levels in mice and humans after radiation-induced mucosal injury and found this conditioning led to an acute VAD state. Consequently, radiation led to a selective loss of both RA-dependent DC subsets and impaired class II-restricted auto and antitumor immunity that could be rescued by supplemental RA. These findings establish a critical role for RA in regulating the homeostasis of pre-DC-derived DC subsets and have implications for the management of patients with immune deficiencies resulting from malnutrition and irradiation.

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The Journal of experimental medicine, 210, 1540-9538, 1961-76, 2013

PMID: 23999499

Open Access

BACH2 represses effector programs to stabilize T(reg)-mediated immune homeostasis.
Roychoudhuri R,Hirahara K,Mousavi K,Clever D,Klebanoff CA,Bonelli M,Sciume G,Zare H,Vahedi G,Dema B,Yu Z,Liu H,Takahashi H,Rao M,Muranski P,Crompton JG,Punkosdy G,Bedognetti D,Wang E,Hoffmann V,Rivera J,Marincola FM,Nakamura A,Sartorelli V,Kanno Y,Gattinoni L,Muto A,Igarashi K,O'Shea JJ,Restifo NP

Through their functional diversification, distinct lineages of CD4(+) T cells can act to either drive or constrain immune-mediated pathology. Transcription factors are critical in the generation of cellular diversity, and negative regulators antagonistic to alternate fates often act in conjunction with positive regulators to stabilize lineage commitment. Genetic polymorphisms within a single locus encoding the transcription factor BACH2 are associated with numerous autoimmune and allergic diseases including asthma, Crohn's disease, coeliac disease, vitiligo, multiple sclerosis and type 1 diabetes. Although these associations point to a shared mechanism underlying susceptibility to diverse immune-mediated diseases, a function for BACH2 in the maintenance of immune homeostasis has not been established. Here, by studying mice in which the Bach2 gene is disrupted, we define BACH2 as a broad regulator of immune activation that stabilizes immunoregulatory capacity while repressing the differentiation programs of multiple effector lineages in CD4(+) T cells. BACH2 was required for efficient formation of regulatory (Treg) cells and consequently for suppression of lethal inflammation in a manner that was Treg-cell-dependent. Assessment of the genome-wide function of BACH2, however, revealed that it represses genes associated with effector cell differentiation. Consequently, its absence during Treg polarization resulted in inappropriate diversion to effector lineages. In addition, BACH2 constrained full effector differentiation within TH1, TH2 and TH17 cell lineages. These findings identify BACH2 as a key regulator of CD4(+) T-cell differentiation that prevents inflammatory disease by controlling the balance between tolerance and immunity.

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Nature, 498, 1476-4687, 506-10, 2013

PMID: 23728300

Open Access

Gene-based vaccination with a mismatched envelope protects against simian immunodeficiency virus infection in nonhuman primates.
Flatz L,Cheng C,Wang L,Foulds KE,Ko SY,Kong WP,Roychoudhuri R,Shi W,Bao S,Todd JP,Asmal M,Shen L,Donaldson M,Schmidt SD,Gall JG,Pinschewer DD,Letvin NL,Rao S,Mascola JR,Roederer M,Nabel GJ

The RV144 trial demonstrated that an experimental AIDS vaccine can prevent human immunodeficiency virus type 1 (HIV-1) infection in humans. Because of its limited efficacy, further understanding of the mechanisms of preventive AIDS vaccines remains a priority, and nonhuman primate (NHP) models of lentiviral infection provide an opportunity to define immunogens, vectors, and correlates of immunity. In this study, we show that prime-boost vaccination with a mismatched SIV envelope (Env) gene, derived from simian immunodeficiency virus SIVmac239, prevents infection by SIVsmE660 intrarectally. Analysis of different gene-based prime-boost immunization regimens revealed that recombinant adenovirus type 5 (rAd5) prime followed by replication-defective lymphocytic choriomeningitis virus (rLCMV) boost elicited robust CD4 and CD8 T-cell and humoral immune responses. This vaccine protected against infection after repetitive mucosal challenge with efficacies of 82% per exposure and 62% cumulatively. No effect was seen on viremia in infected vaccinated monkeys compared to controls. Protection correlated with the presence of neutralizing antibodies to the challenge viruses tested in peripheral blood mononuclear cells. These data indicate that a vaccine expressing a mismatched Env gene alone can prevent SIV infection in NHPs and identifies an immune correlate that may guide immunogen selection and immune monitoring for clinical efficacy trials.

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Journal of virology, 86, 1098-5514, 7760-70, 2012

PMID: 22593152

Open Access

Th17 cells are long lived and retain a stem cell-like molecular signature.
Muranski P,Borman ZA,Kerkar SP,Klebanoff CA,Ji Y,Sanchez-Perez L,Sukumar M,Reger RN,Yu Z,Kern SJ,Roychoudhuri R,Ferreyra GA,Shen W,Durum SK,Feigenbaum L,Palmer DC,Antony PA,Chan CC,Laurence A,Danner RL,Gattinoni L,Restifo NP

Th17 cells have been described as short lived, but this view is at odds with their capacity to trigger protracted damage to normal and transformed tissues. We report that Th17 cells, despite displaying low expression of CD27 and other phenotypic markers of terminal differentiation, efficiently eradicated tumors and caused autoimmunity, were long lived, and maintained a core molecular signature resembling early memory CD8(+) cells with stem cell-like properties. In addition, we found that Th17 cells had high expression of Tcf7, a direct target of the Wnt and β-catenin signaling axis, and accumulated β-catenin, a feature observed in stem cells. In vivo, Th17 cells gave rise to Th1-like effector cell progeny and also self-renewed and persisted as IL-17A-secreting cells. Multipotency was required for Th17 cell-mediated tumor eradication because effector cells deficient in IFN-γ or IL-17A had impaired activity. Thus, Th17 cells are not always short lived and are a less-differentiated subset capable of superior persistence and functionality.

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Immunity, 35, 1097-4180, 972-85, 2011

PMID: 22177921

Open Access

Permissivity of the NCI-60 cancer cell lines to oncolytic Vaccinia Virus GLV-1h68.
Ascierto ML,Worschech A,Yu Z,Adams S,Reinboth J,Chen NG,Pos Z,Roychoudhuri R,Di Pasquale G,Bedognetti D,Uccellini L,Rossano F,Ascierto PA,Stroncek DF,Restifo NP,Wang E,Szalay AA,Marincola FM

Oncolytic viral therapy represents an alternative therapeutic strategy for the treatment of cancer. We previously described GLV-1h68, a modified Vaccinia Virus with exclusive tropism for tumor cells, and we observed a cell line-specific relationship between the ability of GLV-1h68 to replicate in vitro and its ability to colonize and eliminate tumor in vivo.

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BMC cancer, 11, 1471-2407, 451, 2011

PMID: 22011439

Open Access

Single-cell gene-expression profiling reveals qualitatively distinct CD8 T cells elicited by different gene-based vaccines.
Flatz L,Roychoudhuri R,Honda M,Filali-Mouhim A,Goulet JP,Kettaf N,Lin M,Roederer M,Haddad EK,Sékaly RP,Nabel GJ

CD8 T cells play a key role in mediating protective immunity against selected pathogens after vaccination. Understanding the mechanism of this protection is dependent upon definition of the heterogeneity and complexity of cellular immune responses generated by different vaccines. Here, we identify previously unrecognized subsets of CD8 T cells based upon analysis of gene-expression patterns within single cells and show that they are differentially induced by different vaccines. Three prime-boost vector combinations encoding HIV Env stimulated antigen-specific CD8 T-cell populations of similar magnitude, phenotype, and functionality. Remarkably, however, analysis of single-cell gene-expression profiles enabled discrimination of a majority of central memory (CM) and effector memory (EM) CD8 T cells elicited by the three vaccines. Subsets of T cells could be defined based on their expression of Eomes, Cxcr3, and Ccr7, or Klrk1, Klrg1, and Ccr5 in CM and EM cells, respectively. Of CM cells elicited by DNA prime-recombinant adenoviral (rAd) boost vectors, 67% were Eomes(-) Ccr7(+) Cxcr3(-), in contrast to only 7% and 2% stimulated by rAd5-rAd5 or rAd-LCMV, respectively. Of EM cells elicited by DNA-rAd, 74% were Klrk1(-) Klrg1(-)Ccr5(-) compared with only 26% and 20% for rAd5-rAd5 or rAd5-LCMV. Definition by single-cell gene profiling of specific CM and EM CD8 T-cell subsets that are differentially induced by different gene-based vaccines will facilitate the design and evaluation of vaccines, as well as enable our understanding of mechanisms of protective immunity.

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Proceedings of the National Academy of Sciences of the United States of America, 108, 1091-6490, 5724-9, 2011

PMID: 21422297

Open Access

Season of cancer diagnosis exerts distinct effects upon short- and long-term survival.
Roychoudhuri R,Robinson D,Coupland V,Holmberg L,Moller H

Several epidemiological studies have shown an association between the season in which certain cancers are diagnosed and survival, with diagnosis in summer and autumn being associated with better survival. In this study, we have added resolution to the analysis of seasonality in cancer survival by considering mortality within several nonoverlapping time periods following diagnosis, thereby quantifying the separate contributions of mechanisms operating in the short term and in the longer term. We found evidence of seasonality acting on mortality within 2 distinct periods following diagnosis. Diagnosis in the summer was associated with substantially decreased mortality within the first month of diagnosis compared with winter in men with prostate cancer, those of both sexes with colorectal or lung cancer, and most strikingly, amongst women with breast cancer (hazard ratio 0.81 [95% confidence interval 0.75-0.86]). Adjusting for monthly variations in general mortality greatly attenuated the seasonal effects on short-term mortality. At long-term follow-up (>5 years), there was a consistent shift in the seasonality pattern, with autumn diagnosis alone being associated with decreased mortality, both in female breast cancer cases and in lung cancer cases of both sexes. We conclude that the higher survival observed amongst patients diagnosed in summer and autumn is predominantly a short-term phenomenon that is largely attributable to generally higher mortality in winter. However, the distinct mortality reduction observed in the long term amongst those diagnosed in the autumn, especially amongst breast cancer patients, may indicate the presence of a seasonally variable protective mechanism.

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International journal of cancer. Journal international du cancer, 124, 1097-0215, 2436-41, 2009

PMID: 19165867

Increased cardiovascular mortality more than fifteen years after radiotherapy for breast cancer: a population-based study.
Roychoudhuri R,Robinson D,Putcha V,Cuzick J,Darby S,Moller H

Breast radiotherapy as practised in the 1970s and 1980s resulted in significant myocardial exposure, and this was higher when the left breast was treated. It has been proposed that this difference might result in greater cardiovascular mortality following irradiation of the left breast when compared with the right.

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BMC cancer, 7, 1471-2407, 9, 2007

PMID: 17224064

Open Access

Cancer survival is dependent on season of diagnosis and sunlight exposure.
Lim HS,Roychoudhuri R,Peto J,Schwartz G,Baade P,Moller H

Sunlight is essential for the production of vitamin D in the body. Evidence exists to suggest that vitamin D metabolites may have a role in tumor growth suppression. In this large study, involving over a million cancer patients from the United Kingdom, we have analyzed the role of season of diagnosis and sunlight exposure in cancer survival for cancers of the breast, colorectum, lung, prostate and at all sites combined. We used population-based data from the Thames Cancer Registry to analyze cancer survival in periods 0-1 and 0-5 years after diagnosis. The analysis was performed using Cox proportional regression analysis adjusting for age and period at diagnosis and including season of diagnosis and sunlight exposure in the preceding months as factors in the analysis. We found evidence of substantial seasonality in cancer survival, with diagnosis in summer and autumn associated with improved survival compared with that in winter, especially in female breast cancer patients and both male and female lung cancer patients (hazard ratios 0.86 [95% CI 0.83-0.89], 0.95 [95% CI 0.92-0.97] and 0.95 [95% CI 0.93-0.98] respectively). Cumulative sunlight exposure in the months preceding diagnosis was also a predictor of subsequent survival, although season of diagnosis was a stronger predictor than cumulative sunlight exposure. We found seasonality in cancer survival to be stronger in women than in men. Our results add to a growing body of evidence that vitamin D metabolites play an important role in cancer survival.

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International journal of cancer. Journal international du cancer, 119, 0020-7136, 1530-6, 2006

PMID: 16671100

Cancer and laterality: a study of the five major paired organs (UK).
Roychoudhuri R,Putcha V,Moller H

The human body displays marked asymmetry: paired organs differ bilaterally exerting effects upon cancer incidence and progression. However the factors involved remain contentious. In this large study involving over a quarter of a million cancer patients, we examine the epidemiological correlates of cancer laterality including incidence, stage at diagnosis and survival in the five major paired organs: the breasts, lungs, kidneys, testes and ovaries.

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Cancer causes & control : CCC, 17, 0957-5243, 655-62, 2006

PMID: 16633912

Radiation-induced malignancies following radiotherapy for breast cancer.
Roychoudhuri R,Evans H,Robinson D,Moller H

With advances in diagnosis and treatment, breast cancer is becoming an increasingly survivable disease resulting in a large population of long-term survivors. Factors affecting the quality of life of such patients include the consequences of breast cancer treatment, which may have involved radiotherapy. In this study, we compare the incidence of second primary cancers in women who received breast radiotherapy with that in those who did not (non-radiotherapy). All women studied received surgery for their first breast cancer. Second cancers of the lung, colon, oesophagus and thyroid gland, malignant melanomas, myeloid leukaemias and second primary breast cancers were studied. Comparing radiotherapy and non-radiotherapy cohorts, elevated relative risks (RR) were observed for lung cancer at 10-14 years and 15 or more (15+) years after initial breast cancer diagnosis (RR 1.62, 95% confidence interval [CI] 1.05-2.54 and RR 1.49, 95% CI 1.05-2.14, respectively), and for myeloid leukaemia at 1-5 years (RR 2.99, 95% CI 1.13-9.33), for second breast cancer at 5-10 years (RR 1.34, 95% CI 1.10-1.63) and 15+ years (RR 1.26, 95% CI 1.00-1.59) and oesophageal cancer at 15+ years (RR 2.19, 95% CI 1.10-4.62).

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British journal of cancer, 91, 0007-0920, 868-72, 2004

PMID: 15292931

Open Access