Geetha H. Mylvaganam

Efficacy of Metreleptin in Obese Patients With Type 2 Diabetes: Cellular and Molecular Pathways Underlying Leptin Tolerance

OBJECTIVE Metreleptin has been efficacious in improving metabolic control in patients with lipodystrophy, but its efficacy has not been tested in obese patients with type 2 diabetes. RESEARCH DESIGN AND METHODS We studied the role of leptin in regulating the endocrine adaptation to long-term caloric deprivation and weight loss in obese diabetic subjects over 16 weeks in the context of a double-blinded, placebo–controlled, randomized trial. We then performed detailed interventional and mechanistic signaling studies in humans in vivo, ex vivo, and in vitro. RESULTS In obese patients with diabetes, metreleptin administration for 16 weeks did not alter body weight or circulating inflammatory markers but reduced HbA1c marginally (8.01 ± 0.93–7.96 ± 1.12, P = 0.03). Total leptin, leptin-binding protein, and antileptin antibody levels increased, limiting free leptin availability and resulting in circulating free leptin levels of ∼50 ng/mL. Consistent with clinical observations, all metreleptin signaling pathways studied in human adipose tissue and peripheral blood mononuclear cells were saturable at ∼50 ng/mL, with no major differences in timing or magnitude of leptin-activated STAT3 phosphorylation in tissues from male versus female or obese versus lean humans in vivo, ex vivo, or in vitro. We also observed for the first time that endoplasmic reticulum (ER) stress in human primary adipocytes inhibits leptin signaling. CONCLUSIONS In obese patients with diabetes, metreleptin administration did not alter body weight or circulating inflammatory markers but reduced HbA1c marginally. ER stress and the saturable nature of leptin signaling pathways play a key role in the development of leptin tolerance in obese patients with diabetes.

Induction of Th1-Biased T Follicular Helper (Tfh) Cells in Lymphoid Tissues during Chronic Simian Immunodeficiency Virus Infection Defines Functionally Distinct Germinal Center Tfh Cells

Chronic HIV infection is associated with accumulation of germinal center (GC) T follicular helper (Tfh) cells in the lymphoid tissue. The GC Tfh cells can be heterogeneous based on the expression of chemokine receptors associated with T helper lineages, such as CXCR3 (Th1), CCR4 (Th2), and CCR6 (Th17). However, the heterogeneous nature of GC Tfh cells in the lymphoid tissue and its association with viral persistence and Ab production during chronic SIV/HIV infection are not known. To address this, we characterized the expression of CXCR3, CCR4, and CCR6 on GC Tfh cells in lymph nodes following SIVmac251 infection in rhesus macaques. In SIV-naive rhesus macaques, only a small fraction of GC Tfh cells expressed CXCR3, CCR4, and CCR6. However, during chronic SIV infection, the majority of GC Tfh cells expressed CXCR3, whereas the proportion of CCR4+ cells did not change, and CCR6+ cells decreased. CXCR3+, but not CXCR3−, GC Tfh cells produced IFN-γ (Th1 cytokine) and IL-21 (Tfh cytokine), whereas both subsets expressed CD40L following stimulation. Immunohistochemistry analysis demonstrated an accumulation of CD4+IFN-γ+ T cells within the hyperplastic follicles during chronic SIV infection. CXCR3+ GC Tfh cells also expressed higher levels of ICOS, CCR5, and α4β7 and contained more copies of SIV DNA compared with CXCR3− GC Tfh cells. However, CXCR3+ and CXCR3− GC Tfh cells delivered help to B cells in vitro for production of IgG. These data demonstrate that chronic SIV infection promotes expansion of Th1-biased GC Tfh cells, which are phenotypically and functionally distinct from conventional GC Tfh cells and contribute to hypergammaglobulinemia and viral reservoirs.

Leptin administration to overweight and obese subjects for 6 months increases free leptin concentrations but does not alter circulating hormones of the thyroid and IGF axes during weight loss induced by a mild hypocaloric diet

OBJECTIVE Short-term energy deprivation reduces leptin concentrations and alters the levels of circulating hormones of the hypothalamic-pituitary-peripheral axis in lean subjects. Whether the reduction in leptin concentration during long-term weight loss in obese individuals is linked to the same neuroendocrine changes seen in lean, leptin-sensitive subjects remains to be fully clarified. METHODS In this study, 24 overweight and obese adults (16 women and eight men; body mass index (BMI): 27.5-38.0 kg/m(2)) were prescribed a hypocaloric diet (-500 kcal/day) and were randomized to receive recombinant methionyl leptin (n=18, metreleptin, 10 mg/day self-injected s.c.) or placebo (n=6, same volume and time as metreleptin) for 6 months. RESULTS Metreleptin administration did not affect weight loss beyond that induced by hypocaloric diet alone (P for interaction=0.341) but increased the serum concentrations of total leptin by six- to eight-fold (P<0.001) and led to the generation of anti-leptin antibodies. Despite free leptin concentration (P for interaction=0.041) increasing from 9±1 ng/ml at baseline to 43±15 and 36±12 ng/ml at 3 and 6 months, respectively, changes in circulating hormones of the thyroid and IGF axes at 3 and 6 months were not significantly different in the placebo- and metreleptin-treated groups. CONCLUSIONS Leptin does not likely mediate changes in neuroendocrine function in response to weight loss induced by a mild hypocaloric diet in overweight and obese subjects.

Diminished viral control during simian immunodeficiency virus infection is associated with aberrant PD-1hi CD4 T cell enrichment in the lymphoid follicles of the rectal mucosa.

The inhibitory receptor programmed death-1 (PD-1) has been shown to regulate CD8 T cell function during chronic SIV infection; however, its role on CD4 T cells, specifically in the gut-associated lymphoid tissue, is less well understood. In this study, we show that a subset of CD4 T cells expresses high levels of PD-1 (PD-1hi) in the rectal mucosa, a preferential site of virus replication. The majority of these PD-1hi CD4 T cells expressed Bcl-6 and CXCR5, markers characteristic of T follicular helper cells in the lymph nodes. Following a pathogenic SIV infection, the frequency of PD-1hi cells (as a percentage of CD4 T cells) dramatically increased in the rectal mucosa; however, a significant fraction of them did not express CXCR5. Furthermore, only a small fraction of PD-1hi cells expressed CCR5, and despite this low level of viral coreceptor expression, a significant fraction of these cells were productively infected. Interestingly, vaccinated SIV controllers did not present with this aberrant PD-1hi CD4 T cell enrichment, and this lack of enrichment was associated with the presence of higher frequencies of SIV-specific granzyme B+ CD8 T cells within the lymphoid tissue, suggesting a role for antiviral CD8 T cells in limiting aberrant expansion of PD-1hi CD4 T cells. These results highlight the importance of developing vaccines that enhance antiviral CD8 T cells at sites of preferential viral replication and support the need for developing therapeutic interventions that limit expansion of SIV+PD-1hi CD4 T cells at mucosal sites as a means to enhance viral control.

Diminished Viral Control during Simian Immunodeficiency Virus Infection Is Associated with Aberrant PD-1 hi CD4 T Cell Enrichment in the Lymphoid Follicles of the Rectal Mucosa

The inhibitory receptor programmed death-1 (PD-1) has been shown to regulate CD8 T cell function during chronic SIV infection; however, its role on CD4 T cells, specifically in the gut-associated lymphoid tissue, is less well understood. In this study, we show that a subset of CD4 T cells expresses high levels of PD-1 (PD-1hi) in the rectal mucosa, a preferential site of virus replication. The majority of these PD-1hi CD4 T cells expressed Bcl-6 and CXCR5, markers characteristic of T follicular helper cells in the lymph nodes. Following a pathogenic SIV infection, the frequency of PD-1hi cells (as a percentage of CD4 T cells) dramatically increased in the rectal mucosa; however, a significant fraction of them did not express CXCR5. Furthermore, only a small fraction of PD-1hi cells expressed CCR5, and despite this low level of viral coreceptor expression, a significant fraction of these cells were productively infected. Interestingly, vaccinated SIV controllers did not present with this aberrant PD-1hi CD4 T cell enrichment, and this lack of enrichment was associated with the presence of higher frequencies of SIV-specific granzyme B+ CD8 T cells within the lymphoid tissue, suggesting a role for antiviral CD8 T cells in limiting aberrant expansion of PD-1hi CD4 T cells. These results highlight the importance of developing vaccines that enhance antiviral CD8 T cells at sites of preferential viral replication and support the need for developing therapeutic interventions that limit expansion of SIV+PD-1hi CD4 T cells at mucosal sites as a means to enhance viral control.

Circulating adiponectin is inversely associated with risk of thyroid cancer: in vivo and in vitro studies.

CONTEXT Circulating adiponectin has been inversely associated with risk for several malignancies. Its association with thyroid cancer has not yet been evaluated. OBJECTIVE/METHODS We measured circulating adiponectin levels in 175 thyroid carcinoma patients and 107 controls. We also examined the expression of adiponectin receptors (AdipoR1 and AdipoR2) using immunohistochemistry in 82 thyroid carcinoma tissues and using RT-qPCR in 40 human thyroid carcinoma tissues (32 papillary, six follicular/Hurthle, one anaplastic, one medullary), four normal human thyroid tissue specimens, and the BHP7 and SW579 thyroid cancer cell lines. We then utilized these thyroid cancer cell lines to investigate whether adiponectin could directly regulate cell cycle or apoptosis. RESULTS Thyroid cancer patients had lower circulating adiponectin levels than controls (17.00 ± 6.32 vs. 19.26 ± 6.28 μg/ml; P < 0.001). Subjects in the highest tertile of circulating adiponectin concentrations had significantly lower odds of developing any type of thyroid carcinoma (odds ratio = 0.29; 95% confidence interval, 0.16-0.55), or papillary thyroid carcinoma (odds ratio = 0.27; 95% confidence interval, 0.14-0.55), before and after adjustment for potential confounders. Both thyroid carcinoma cell lines and tissues expressed AdipoR1 and AdipoR2. Recombinant adiponectin did not exert a clinically significant direct effect on cell cycle, proliferation, or apoptosis in thyroid cancer cell lines in vitro. CONCLUSIONS Circulating adiponectin is independently and inversely associated with the risk of thyroid cancer. Human thyroid carcinomas and cell lines express adiponectin receptors. However, in the absence of a major direct effect of adiponectin on thyroid cancer cell lines in vitro, the negative association observed herein may be attributed to the metabolic effects of adiponectin.

Dynamics of SIV-specific CXCR5+CD8 T cells during chronic SIV infection

Significance Simian immunodeficiency virus (SIV)-specific follicular CD8 T cells represent a unique subset of antiviral CD8 T cells that rapidly expand during pathogenic SIV infection, localize within B-cell follicles, and contribute to control of chronic SIV replication. The potential for these cells to infiltrate sites of ongoing viral replication and viral persistence and the ability to induce these cells by vaccination provide a tremendous opportunity to develop and optimize therapeutic strategies to target and reduce the HIV reservoirs in lymphoid tissues. A significant challenge to HIV eradication is the elimination of viral reservoirs in germinal center (GC) T follicular helper (Tfh) cells. However, GCs are considered to be immune privileged for antiviral CD8 T cells. Here, we show a population of simian immunodeficiency virus (SIV)-specific CD8 T cells express CXCR5 (C-X-C chemokine receptor type 5, a chemokine receptor required for homing to GCs) and expand in lymph nodes (LNs) following pathogenic SIV infection in a cohort of vaccinated macaques. This expansion was greater in animals that exhibited superior control of SIV. The CXCR5+ SIV-specific CD8 T cells demonstrated enhanced polyfunctionality, restricted expansion of antigen-pulsed Tfh cells in vitro, and possessed a unique gene expression pattern related to Tfh and Th2 cells. The increase in CXCR5+ CD8 T cells was associated with the presence of higher frequencies of SIV-specific CD8 T cells in the GC. Following TCR-driven stimulation in vitro, CXCR5+ but not CXCR5– CD8 T cells generated both CXCR5+ as well as CXCR5– cells. However, the addition of TGF-β to CXCR5– CD8 T cells induced a population of CXCR5+ CD8 T cells, suggesting that this cytokine may be important in modulating these CXCR5+ CD8 T cells in vivo. Thus, CXCR5+ CD8 T cells represent a unique subset of antiviral CD8 T cells that expand in LNs during chronic SIV infection and may play a significant role in the control of pathogenic SIV infection.

Minor Antigens on Transfused RBCs Crossprime CD8 T Cells but Do Not Induce Full Effector Function

HLA‐matched bone marrow transplantation (BMT) is a cure for nonmalignant hematological disorders; however, rejection rates are high and correlate with the number of antecedent transfusions. Recently, using murine models, we reported that minor antigens (mHAs) in transfused leukoreduced red blood cell (RBC) or platelet units induce rejection of subsequent BMT. To study RBCs as an immunogen, we utilized transgenic donors that express a model mHA selectively on RBCs (HOD mouse). Transfusion of HOD blood did not induce BMT rejection of marrow that shared mHAs with the HOD RBCs. Similarly, no endogenous anti‐HOD CD8+ T‐cell response was detected with antigen‐specific tetramer reagents. Adoptively transferred OT‐I T cells rapidly expanded after HOD blood transfusion; however, only a semi‐effector phenotype was observed (tumor necrosis factor‐α and interferon‐γ secretion, but essentially no Granzyme B). After initial expansion, OT‐I T cells contracted rapidly to very low levels. A similar trend was observed by in vivo CTL assay, with only transient lytic activity. Together, these data indicate that RBCs may not be the component of RBC units that induces BMT rejection, and suggest that contaminating platelets or leukocytes may be responsible.

Combination anti–PD-1 and antiretroviral therapy provides therapeutic benefit against SIV

Therapeutic strategies that augment antiviral immunity and reduce the viral reservoir are critical to achieving durable remission of HIV. The coinhibitory receptor programmed death-1 (PD-1) regulates CD8+ T cell dysfunction during chronic HIV and SIV infections. We previously demonstrated that in vivo blockade of PD-1 during chronic SIV infection improves the function of antiviral CD8+ T cells and B cells. Here, we tested the immunological and virological effects of PD-1 blockade combined with antiretroviral therapy (ART) in rhesus macaques. Administration of anti-PD-1 antibody 10 days prior to ART initiation rapidly enhanced antiviral CD8+ T cell function and diminished IFN-stimulated genes. This resulted in faster viral suppression in plasma and better Th17 cell reconstitution in the rectal mucosa following ART initiation. PD-1 blockade during ART resulted in lower levels of cell-associated replication-competent virus. Following ART interruption, PD-1 antibody-treated animals showed markedly higher expansion of proliferating CXCR5+perforin+granzyme B+ effector CD8+ T cells and lower regulatory T cells that resulted in better control of viremia. Our results show that PD-1 blockade can be administered safely with ART to augment antiviral CD8+ T cell function and reduce the viral reservoir, leading to improved control of viral rebound after ART interruption.

Tfh1 Cells in Germinal Centers During Chronic HIV/SIV Infection

T follicular helper CD4 cells (Tfh) are essential for the development and maintenance of germinal center (GC) reactions, a critical process that promotes the generation of long-lived high affinity humoral immunity. It is becoming increasingly evident that GC-Tfh cells are heterogeneous in nature with some cellular characteristics associated with a Th1, Th2, and Th17 phenotype. Emerging studies suggest that GC-Tfh cells are directed to differentiate into distinct phenotypes during chronic HIV/SIV infection and these changes in GC-Tfh cells can greatly impact the B cell response and subclass of antibodies generated. Studies in HIV-infected humans have shown that certain Tfh phenotypes are associated with the generation of broadly neutralizing antibody responses. Moreover, the susceptibility of particular GC-Tfh subsets to HIV infection within the secondary lymphoid sites can also impact GC-Tfh/B cell interactions. In this review, we discuss the recent advances that show Tfh heterogeneity during chronic HIV/SIV infection. In particular, we will discuss the dynamics of GC-Tfh cells, their altered differentiation state and function, and their impact on B cell responses during HIV/SIV infection. In addition, we will also discuss the potential role of a recently described novel subset of follicular homing CXCR5+ CD8 T cells (Tfc) and their importance in contributing to control of chronic HIV/SIV infection. A better understanding of the mechanistic role of follicular homing CD4 and CD8 T cells during HIV/SIV infection will aid in the design of vaccines and therapeutic strategies to prevent and treat HIV/AIDS.