T-Cell Acute Lymphoblastic Leukemia/Lymphoma (T-ALL) With Negative Screening Immaturity Markers and Gamma-Delta Receptor Expression.

T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) is characterized by the combination of T-cell lineage and the presence of immaturity marker(s). Sometimes, the most common immaturity markers for initial flow cytometry screening in T-ALL may be negative, which can be a diagnostic pitfall. When a lack of common first-line immaturity markers is encountered in combination with gamma/delta T-cell receptor expression, a misdiagnosis of mature gamma-delta T-cell leukemia/lymphoma could be rendered. Here, we discuss two T-ALL cases with the absence of common flow cytometry immaturity markers and positive gamma/delta receptor expression.

Transcriptional and Immunologic Correlates of Response to Pandemic Influenza Vaccine in Aviremic, HIV-Infected Children.

People living with HIV (PWH) often exhibit poor responses to influenza vaccination despite effective combination anti-retroviral (ART) mediated viral suppression. There exists a paucity of data in identifying immune correlates of influenza vaccine response in context of HIV infection that would be useful in improving its efficacy in PWH, especially in younger individuals. Transcriptomic data were obtained by microarray from whole blood isolated from aviremic pediatric and adolescent HIV-infected individuals (4-25 yrs) given two doses of Novartis/H1N1 09 vaccine during the pandemic H1N1 influenza outbreak. Supervised clustering and gene set enrichment identified contrasts between individuals exhibiting high and low antibody responses to vaccination. High responders exhibited hemagglutination inhibition antibody titers >1:40 post-first dose and 4-fold increase over baseline. Baseline molecular profiles indicated increased gene expression in metabolic stress pathways in low responders compared to high responders. Inflammation-related and interferon-inducible gene expression pathways were higher in low responders 3 wks post-vaccination. The broad age range and developmental stage of participants in this study prompted additional analysis by age group (e.g. <13yrs and ≥13yrs). This analysis revealed differential enrichment of gene pathways before and after vaccination in the two age groups. Notably, CXCR5, a homing marker expressed on T follicular helper (Tfh) cells, was enriched in high responders (>13yrs) following vaccination which was accompanied by peripheral Tfh expansion. Our results comprise a valuable resource of immune correlates of vaccine response to pandemic influenza in HIV infected children that may be used to identify favorable targets for improved vaccine design in different age groups.

p53-inducible SESTRINs might play opposite roles in the regulation of early and late stages of lung carcinogenesis.

SESTRINs (SESN1-3) are proteins encoded by an evolutionarily conserved gene family that plays an important role in the regulation of cell viability and metabolism in response to stress. Many of the effects of SESTRINs are mediated by negative and positive regulation of mechanistic target of rapamycin kinase complexes 1 and 2 (mTORC1 and mTORC2), respectively, that are often deregulated in human cancers where they support cell growth, proliferation, and cell viability. Besides their effects on regulation of mTORC1/2, SESTRINs also control the accumulation of reactive oxygen species, cell death, and mitophagy. SESN1 and SESN2 are transcriptional targets of tumor suppressor protein p53 and may mediate tumor suppressor activities of p53. Therefore, we conducted studies based on a mouse lung cancer model and human lung adenocarcinoma A549 cells to evaluate the potential impact of SESN1 and SESN2 on lung carcinogenesis. While we observed that expression of SESN1 and SESN2 is often decreased in human tumors, inactivation of Sesn2 in mice positively regulates tumor growth through a mechanism associated with activation of AKT, while knockout of Sesn1 has no additional impact on carcinogenesis in Sesn2-deficient mice. However, inactivation of SESN1 and/or SESN2 in A549 cells accelerates cell proliferation and imparts resistance to cell death in response to glucose starvation. We propose that despite their contribution to early tumor growth, SESTRINs might suppress late stages of carcinogenesis through inhibition of cell proliferation or activation of cell death in conditions of nutrient deficiency.

Genetic and epigenetic inactivation of SESTRIN1 controls mTORC1 and response to EZH2 inhibition in follicular lymphoma.

Follicular lymphoma (FL) is an incurable form of B cell lymphoma. Genomic studies have cataloged common genetic lesions in FL such as translocation t(14;18), frequent losses of chromosome 6q, and mutations in epigenetic regulators such as EZH2 Using a focused genetic screen, we identified SESTRIN1 as a relevant target of the 6q deletion and demonstrate tumor suppression by SESTRIN1 in vivo. Moreover, SESTRIN1 is a direct target of the lymphoma-specific EZH2 gain-of-function mutation (EZH2Y641X ). SESTRIN1 inactivation disrupts p53-mediated control of mammalian target of rapamycin complex 1 (mTORC1) and enables mRNA translation under genotoxic stress. SESTRIN1 loss represents an alternative to RRAGC mutations that maintain mTORC1 activity under nutrient starvation. The antitumor efficacy of pharmacological EZH2 inhibition depends on SESTRIN1, indicating that mTORC1 control is a critical function of EZH2 in lymphoma. Conversely, EZH2Y641X mutant lymphomas show increased sensitivity to RapaLink-1, a bifunctional mTOR inhibitor. Hence, SESTRIN1 contributes to the genetic and epigenetic control of mTORC1 in lymphoma and influences responses to targeted therapies.

Sestrin2 is induced by glucose starvation via the unfolded protein response and protects cells from non-canonical necroptotic cell death.

Sestrin2 is a member of a family of stress responsive proteins, which controls cell viability via antioxidant activity and regulation of the mammalian target of rapamycin protein kinase (mTOR). Sestrin2 is induced by different stress insults, which diminish ATP production and induce energetic stress in the cells. Glucose is a critical substrate for ATP production utilized via glycolysis and mitochondrial respiration as well as for glycosylation of newly synthesized proteins in the endoplasmic reticulum (ER) and Golgi. Thus, glucose starvation causes both energy deficiency and activation of ER stress followed by the unfolding protein response (UPR). Here, we show that UPR induces Sestrin2 via ATF4 and NRF2 transcription factors and demonstrate that Sestrin2 protects cells from glucose starvation-induced cell death. Sestrin2 inactivation sensitizes cells to necroptotic cell death that is associated with a decline in ATP levels and can be suppressed by Necrostatin 7. We propose that Sestrin2 protects cells from glucose starvation-induced cell death via regulation of mitochondrial homeostasis.

Sestrin2 facilitates death receptor-induced apoptosis in lung adenocarcinoma cells through regulation of XIAP degradation.

Apoptosis plays a critical physiological role in controlling cell number and eliminating damaged, non-functional and transformed cells. Cancerous cells as well as some types of normal cells are often resistant to cell death induced by pro-inflammatory cytokines through death receptors. This potentially allows cancer cells to evade the control from the immune system and to proceed toward a more malignant stage, although the mechanisms of this evasion are not well established. We have recently identified the stress-responsive Sestrin2 protein as a critical regulator of cell viability under stress conditions. Sestrin2 is a member of a small family of antioxidant proteins and inhibitors of mechanistic Target of Rapamycin Complex 1 (mTORC1) kinase. Down-regulation of Sestrin1/2 leads to genetic instability and accelerates the growth of lung adenocarcinoma xenografts. Here we addressed the potential role of Sestrin2 in regulation of cell death induced by TNFR1 and related Fas and TRAIL receptors in lung adenocarcinoma cells. We found that Sestrin2 silencing strongly inhibits cytokine-induced cell death through a mechanism independent of ROS and mTORC1 regulation. We determined that the X-linked inhibitor of apoptosis protein (XIAP) plays a critical role in the control of cytokine-induced cell death by Sestrin2. Thus our study defines a new, previously unrecognized role of Sestrin2 in the regulation of apoptosis.

HIV infection Worsens Age-Associated Defects in Antibody Responses to Influenza Vaccine.

BACKGROUND: Antibody responses to seasonal influenza vaccines are defective during older age and human immunodeficiency virus (HIV) infection. The effect of HIV on immune function in aging is relatively unknown. METHODS: HIV-infected and HIV-uninfected young women (age, 19-54 years) and older women (age, >55 years) were evaluated for B-cell and T-cell responses before and 4 weeks after influenza vaccination. RESULTS: Frequencies of seroprotection pre-vaccination and vaccine responsiveness (≥4-fold increase in antibody titer) were lower in HIV-infected participants than in age-matched HIV-uninfected participants. A subgroup of vaccine nonresponders were compared to responders and found to have reduced frequencies of memory B cells and antigen-specific antibody-secreting cells after vaccination. Frequencies of peripheral T-follicular helper (pTfh) cells correlated with memory B-cell function and influenza A(H1N1) antibody titers. Serologic and immunologic deficits were most frequent in older HIV-infected participants. Underlying CD4(+) T-cell immune activation and inflammation correlated negatively with antibody titers and B-cell function, which was not enhanced by exogenous interleukin 21 supplementation in HIV-infected, older vaccine nonresponders. CONCLUSIONS: Immune activation associated with HIV infection and impaired pTfh function heighten deficiencies in antibody responses to influenza vaccine in older individuals. Strategies to reduce immune activation or augment pTfh function may enhance antibody responses in the aging HIV-infected population.

Sestrins inhibit mTORC1 kinase activation through the GATOR complex.

The mechanistic target of rapamycin complex 1 (mTORC1) kinase is a sensor of different environmental conditions and regulator of cell growth, metabolism, and autophagy. mTORC1 is activated by Rag GTPases, working as RagA:RagB and RagC:RagD heterodimers. Rags control mTORC1 activity by tethering mTORC1 to the lysosomes where it is activated by Rheb GTPase. RagA:RagB, active in its GTP-bound form, is inhibited by GATOR1 complex, a GTPase-activating protein, and GATOR1 is in turn negatively regulated by GATOR2 complex. Sestrins are stress-responsive proteins that inhibit mTORC1 via activation of AMP-activated protein kinase (AMPK) and tuberous sclerosis complex. Here we report an AMPK-independent mechanism of mTORC1 inhibition by Sestrins mediated by their interaction with GATOR2. As a result of this interaction, the Sestrins suppress mTOR lysosomal localization in a Rag-dependent manner. This mechanism is potentially involved in mTORC1 regulation by amino acids, rotenone, and tunicamycin, connecting stress response with mTORC1 inhibition.

Impaired antibody response to influenza vaccine in HIV-infected and uninfected aging women is associated with immune activation and inflammation.

BACKGROUND: Aging and HIV infection are independently associated with excessive immune activation and impaired immune responses to vaccines, but their relationships have not been examined. METHODS: For selecting an aging population we enrolled 28 post-menopausal women including 12 healthy volunteers and 16 HIV-infected women on antiretroviral treatment with <100 HIV RNA copies/ml. Antibody titers to trivalent influenza vaccination given during the 2011-2012 season were determined before and 4 weeks after vaccination. RESULTS: Seroprotective influenza antibody titers (≥ 1:40) were observed in 31% HIV(+) and 58% HIV-uninfected women pre-vaccination. Following vaccination, magnitude of antibody responses and frequency of seroprotection were lower in HIV(+) (75%) than in HIV(-) (91%) women. Plasma IL-21, the signature cytokine of T follicular helper cells (Tfh), and CD4 T cell IL-21R were upregulated with seroconversion (≥ 4 fold increase in antibody titer). Post-vaccine antibody responses were inversely correlated with pre-vaccination plasma TNFα levels and with activated CD4 T cells, including activated peripheral (p)Tfh. Plasma TNFα levels were correlated with activated pTfh cells (r=0.48, p=0.02), and inversely with the post-vaccination levels of plasma IL-21 (r=-0.53, p=0.02). In vitro TNFα blockade improved the ability of CD4 T cells to produce IL-21 and of B cells to secrete immunoglobulins, and addition of exogenous IL-21 to cell cultures enhanced B cell function. Higher frequencies of activated and exhausted CD8 T and B cells were noted in HIV(+) women, but these markers did not show a correlation with antibody responses. CONCLUSIONS: In aging HIV-infected and uninfected women, activated CD4 and pTfh cells may compromise influenza vaccine-induced antibody response, for which a mechanism of TNFα-mediated impairment of pTfh-induced IL-21 secretion is postulated. Interventions aimed at reducing chronic inflammation and immune activation in aging, HIV-infected patients may improve their response to vaccines.

Immune activation in HIV-infected aging women on antiretrovirals--implications for age-associated comorbidities: a cross-sectional pilot study.

BACKGROUND: Persistent immune activation and microbial translocation associated with HIV infection likely place HIV-infected aging women at high risk of developing chronic age-related diseases. We investigated immune activation and microbial translocation in HIV-infected aging women in the post-menopausal ages. METHODS: Twenty-seven post-menopausal women with HIV infection receiving antiretroviral treatment with documented viral suppression and 15 HIV-negative age-matched controls were enrolled. Levels of immune activation markers (T cell immune phenotype, sCD25, sCD14, sCD163), microbial translocation (LPS) and biomarkers of cardiovascular disease and impaired cognitive function (sVCAM-1, sICAM-1 and CXCL10) were evaluated. RESULTS: T cell activation and exhaustion, monocyte/macrophage activation, and microbial translocation were significantly higher in HIV-infected women when compared to uninfected controls. Microbial translocation correlated with T cell and monocyte/macrophage activation. Biomarkers of cardiovascular disease and impaired cognition were elevated in women with HIV infection and correlated with immune activation. CONCLUSIONS: HIV-infected antiretroviral-treated aging women who achieved viral suppression are in a generalized status of immune activation and therefore are at an increased risk of age-associated end-organ diseases compared to uninfected age-matched controls.

Role of IL-21 and IL-21 receptor on B cells in HIV infection.

Interleukin (IL)-21 is a member of a family of cytokines that includes IL-2, IL-4, IL-7, IL-9, and IL-15, all of which utilize a common γ chain in their individual receptor complexes for delivering intracellular signals in their target cells. IL-21 is produced by CD4+ T-cells, in particular follicular T-helper cells, and is critically important in the regulation and maintenance of T cells and B cells in innate and adaptive immunity. The effects of IL-21 are pleiotropic because of the broad cellular distribution of the IL-21 receptor, and it plays a critical role in T cell-dependent and -independent human B cell differentiation for generating humoral immune responses. This article reviews the current knowledge about the importance of IL-21 and IL-21 receptor interaction in human B cell responses, immune defects of B cells and IL-21 in HIV infection, and the potential applicability of IL-21 in vaccines/immunotherapeutic approaches to augment relevant immune responses.

The role of interleukin-21 in HIV infection.

Interleukin (IL)-21 is one of a group of cytokines including IL-2, IL-4, IL-7, IL-9 and IL-15 whose receptor complexes share the common γ chain (γ(c)). Secretion of IL-21 is restricted mainly to T follicular helper (TFH) CD4 T cell subset with contributions from Th17, natural killer (NK) T cells, but the effects of IL-21 are pleiotropic, owing to the broad cellular distribution of the IL-21 receptor. The role of IL-21 in sustaining and regulating T cell, B cell and NK cell responses during chronic viral infections has recently come into focus. This chapter reviews current knowledge about the biology of IL-21 in the context of HIV infection.

Impaired peripheral blood T-follicular helper cell function in HIV-infected nonresponders to the 2009 H1N1/09 vaccine.

The generation of Ab-secreting plasma cells depends critically on CD4 T-follicular helper (TFH) cells during the germinal center reaction. Germinal center TFH cells share functional properties with circulating CXCR5(+) CD4 T cells, referred to herein as peripheral TFH (pTFH) cells. Because deficient Ab production and CD4 T-cell loss are recognized features of HIV infection, in the present study, we investigated pTFH cells in 25 HIV-infected patients on antiretroviral therapy. pTFH frequency was equivalent in patients and healthy controls (HCs), and these cells displayed a central memory phenotype. Sixteen patients and 8 HCs in this group were given a single dose of H1N1/09 influenza vaccine during the 2009 H1N1 influenza outbreak. In the vaccine responders (n = 8) and HCs, pTFH cells underwent expansion with increased IL-21 and CXCL13 secretion in H1N1-stimulated PBMC culture supernatants at week 4 (T2). These changes were not seen in vaccine nonresponders (n = 8). In coculture experiments, sorted pTFH cells supported HIN1-stimulated IgG production by autologous B cells only in vaccine responders. At T2, frequencies of pTFH were correlated with memory B cells, serum H1N1 Ab titers, and Ag-induced IL-21 secretion. Characterization of pTFH cells may provide additional insight into cellular determinants of vaccine-induced Ab response, which may have relevance for vaccine design.

Synergy of brief activation of CD8 T-cells in the presence of IL-12 and adoptive transfer into lymphopenic hosts promotes tumor clearance and anti-tumor memory.

Adoptive T-cell therapy holds great promise for the treatment of metastatic melanoma. However, prohibitive costs associated with current technology required for culture and expansion of tumor-reactive T-cells, the need for intense preconditioning regimens to induce lymphopenia, and the unpredictable anti-tumor effect of adoptively transferred T-cells remain significant impediments for its clinical implementation. Here we report a simplified combinatorial approach that involves short activation of CD8(+) T cells in the presence of IL-12 followed by adoptive transfer into tumor bearing animals after a single injection of cyclophosphamide. This approach resulted in complete eradication of B16 melanoma, and the establishment of long term immunological memory capable of fully protecting mice after a second B16 melanoma challenge. The activated donor cells were unique because they simultaneously exhibited traits for cytotoxic effector function, central memory-like, homing, and senescence. After tumor eradication and within three months after transfer, CD8+ cells exhibited a conventional memory CTL phenotype. Moreover, these memory CTLs acquired functional attributes characteristic of memory stem cells, including the ability to resist chemotherapy-induced toxicity. Our results suggest that short-term T-cell receptor signaling in the presence of IL-12 promotes promiscuous qualities in naïve CTL which - upon transfer into lymphopenic hosts- are sufficient to eradicate tumors and generate life-long tumor-specific memory.

Interleukin-21 and cellular activation concurrently induce potent cytotoxic function and promote antiviral activity in human CD8 T cells.

Infection with human immunodeficiency virus (HIV)-1 induces a progressive deterioration of the immune system that ultimately leads to acquired immune deficiency syndrome (AIDS). Murine models indicate that the common γ-chain (γ(c))-sharing cytokine interleukin (IL)-21 and its receptor (IL-21R) play a crucial role in maintaining polyfunctional T cell responses during chronic viral infections. Therefore, we analyzed the ability of this cytokine to modulate the properties of human CD8 T cells in comparison with other γ(c)-sharing cytokines (IL-2, IL-7, and IL-15). CD8 T cells from healthy volunteers were stimulated in vitro via T cell receptor signals to mimic the heightened status of immune activation of HIV-infected patients. The administration of IL-21 upregulated cytotoxic effector function and the expression of the costimulatory molecule CD28. Notably, this outcome was not accompanied by increased cellular proliferation or activation. Moreover, IL-21 promoted antiviral activity while not inducing HIV-1 replication in vitro. Thus, IL-21 may be a favorable molecule for immunotherapy and a suitable vaccine adjuvant in HIV-infected individuals.