Maturation Pathway from Germline to Broad HIV-1 Neutralizer of a CD4-Mimic Antibody.

Antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. Here, we define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level of somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. We integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages.

Stochastic Expansions Maintain the Clonal Stability of CD8+ T Cell Populations Undergoing Memory Inflation Driven by Murine Cytomegalovirus.

CMV is an obligate and persistent intracellular pathogen that continually drives the production of highly differentiated virus-specific CD8+ T cells in an Ag-dependent manner, a phenomenon known as memory inflation. Extensive proliferation is required to generate and maintain inflationary CD8+ T cell populations, which are counterintuitively short-lived and typically exposed to limited amounts of Ag during the chronic phase of infection. An apparent discrepancy therefore exists between the magnitude of expansion and the requirement for ongoing immunogenic stimulation. To address this issue, we explored the clonal dynamics of memory inflation. First, we tracked congenically marked OT-I cell populations in recipient mice infected with murine CMV (MCMV) expressing the cognate Ag OVA. Irrespective of numerical dominance, stochastic expansions were observed in each population, such that dominant and subdominant OT-I cells were maintained at stable frequencies over time. Second, we characterized endogenous CD8+ T cell populations specific for two classic inflationary epitopes, M38 and IE3. Multiple clonotypes simultaneously underwent Ag-driven proliferation during latent infection with MCMV. In addition, the corresponding CD8+ T cell repertoires were stable over time and dominated by persistent clonotypes, many of which also occurred in more than one mouse. Collectively, these data suggest that stochastic encounters with Ag occur frequently enough to maintain oligoclonal populations of inflationary CD8+ T cells, despite intrinsic constraints on epitope display at individual sites of infection with MCMV.

Preferential Small Intestine Homing and Persistence of CD8 T Cells in Rhesus Macaques Achieved by Molecularly Engineered Expression of CCR9 and Reduced Ex Vivo Manipulation.

Adoptive cell transfer (ACT) is a powerful experimental approach to directly study T-cell-mediated immunity in vivo In the rhesus macaque AIDS virus model, infusing simian immunodeficiency virus (SIV)-infected animals with CD8 T cells engineered to express anti-SIV T-cell receptor specificities enables direct experimentation to better understand antiviral T-cell immunity in vivo Limiting factors in ACT experiments include suboptimal trafficking to, and poor persistence in, the secondary lymphoid tissues targeted by AIDS viruses. Previously, we redirected CD8 T cells to B-cell follicles by ectopic expression of the CXCR5 homing protein. Here, we modify peripheral blood mononuclear cell (PBMC)-derived CD8 T cells to express the CCR9 chemokine receptor, which induces preferential homing of the engineered cells to the small intestine, a site of intense early AIDS virus replication and pathology in rhesus macaques. Additionally, we increase in vivo persistence and overall systemic distribution of infused CD8 T cells, especially in secondary lymphoid tissues, by minimizing ex vivo culture/manipulation, thereby avoiding the loss of CD28+/CD95+ central memory T cells by differentiation in culture. These proof-of-principle results establish the feasibility of preferentially localizing PBMC-derived CD8 T cells to the small intestine and enables the direct experimental ACT-based assessment of the potential role of the quality and timing of effective antiviral CD8 T-cell responses to inhibit viral infection and subsequent replication in small intestine CD4 T cells. More broadly, these results support the engineered expression of homing proteins to direct CD8 T cells to target tissues as a means for both experimental and potential therapeutic advances in T-cell immunotherapies, including cancer.IMPORTANCEAdoptive cell transfer (ACT) of T cells engineered with antigen-specific effector properties can deliver targeted immune responses against malignancies and infectious diseases. Current T-cell-based therapeutic ACT relies on circulatory distribution to deliver engineered T cells to their targets, an approach which has proven effective for some leukemias but provided only limited efficacy against solid tumors. Here, engineered expression of the CCR9 homing receptor redirected CD8 T cells to the small intestine in rhesus macaque ACT experiments. Targeted homing of engineered T-cell immunotherapies holds promise to increase the effectiveness of adoptively transferred cells in both experimental and clinical settings.

Type I interferon responses in rhesus macaques prevent SIV infection and slow disease progression.

Inflammation in HIV infection is predictive of non-AIDS morbidity and death, higher set point plasma virus load and virus acquisition; thus, therapeutic agents are in development to reduce its causes and consequences. However, inflammation may simultaneously confer both detrimental and beneficial effects. This dichotomy is particularly applicable to type I interferons (IFN-I) which, while contributing to innate control of infection, also provide target cells for the virus during acute infection, impair CD4 T-cell recovery, and are associated with disease progression. Here we manipulated IFN-I signalling in rhesus macaques (Macaca mulatta) during simian immunodeficiency virus (SIV) transmission and acute infection with two complementary in vivo interventions. We show that blockade of the IFN-I receptor caused reduced antiviral gene expression, increased SIV reservoir size and accelerated CD4 T-cell depletion with progression to AIDS despite decreased T-cell activation. In contrast, IFN-α2a administration initially upregulated expression of antiviral genes and prevented systemic infection. However, continued IFN-α2a treatment induced IFN-I desensitization and decreased antiviral gene expression, enabling infection with increased SIV reservoir size and accelerated CD4 T-cell loss. Thus, the timing of IFN-induced innate responses in acute SIV infection profoundly affects overall disease course and outweighs the detrimental consequences of increased immune activation. Yet, the clinical consequences of manipulation of IFN signalling are difficult to predict in vivo and therapeutic interventions in human studies should be approached with caution.

The peripheral differentiation of human natural killer T cells.

The peripheral maturation of human CD1d-restricted natural killer T (NKT) cells has not been well described. In this study, we identified four major subsets of NKT cells in adults, distinguished by the expression of CD4, CD8 and CCR5. Phenotypic analysis suggested a hierarchical pattern of differentiation, whereby immature CD4+ CD8- CCR5- cells progressed to an intermediate CD4+ CD8- CCR5+ stage, which remained less differentiated than the CD4- CD8- and CD4- CD8+ subsets, both of which expressed CCR5. This interpretation was supported by functional data, including clonogenic potential and cytokine secretion profiles, as well as T-cell receptor (TCR) excision circle analysis. Moreover, conventional and high-throughput sequencing of the corresponding TCR repertoires demonstrated significant clonotypic overlap within individuals, especially between the more differentiated CD4- CD8- and CD4- CD8+ subsets. Collectively, these results mapped a linear differentiation pathway across the post-thymic landscape of human CD1d-restricted NKT cells.

Epitope specificity delimits the functional capabilities of vaccine-induced CD8 T cell populations.

Despite progress toward understanding the correlates of protective T cell immunity in HIV infection, the optimal approach to Ag delivery by vaccination remains uncertain. We characterized two immunodominant CD8 T cell populations generated in response to immunization of BALB/c mice with a replication-deficient adenovirus serotype 5 vector expressing the HIV-derived Gag and Pol proteins at equivalent levels. The Gag-AI9/H-2K(d) epitope elicited high-avidity CD8 T cell populations with architecturally diverse clonotypic repertoires that displayed potent lytic activity in vivo. In contrast, the Pol-LI9/H-2D(d) epitope elicited motif-constrained CD8 T cell repertoires that displayed lower levels of physical avidity and lytic activity despite equivalent measures of overall clonality. Although low-dose vaccination enhanced the functional profiles of both epitope-specific CD8 T cell populations, greater polyfunctionality was apparent within the Pol-LI9/H-2D(d) specificity. Higher proportions of central memory-like cells were present after low-dose vaccination and at later time points. However, there were no noteworthy phenotypic differences between epitope-specific CD8 T cell populations across vaccine doses or time points. Collectively, these data indicate that the functional and phenotypic properties of vaccine-induced CD8 T cell populations are sensitive to dose manipulation, yet constrained by epitope specificity in a clonotype-dependent manner.

Magnitude of daily energy deficit predicts frequency but not severity of menstrual disturbances associated with exercise and caloric restriction.

We assessed the impact of energy deficiency on menstrual function using controlled feeding and supervised exercise over four menstrual cycles (1 baseline and 3 intervention cycles) in untrained, eumenorrheic women aged 18-30 yr. Subjects were randomized to either an exercising control (EXCON) or one of three exercising energy deficit (ED) groups, i.e., mild (ED1; -8 ± 2%), moderate (ED2; -22 ± 3%), or severe (ED3; -42 ± 3%). Menstrual cycle length and changes in urinary concentrations of estrone-1-glucuronide, pregnanediol glucuronide, and midcycle luteinizing hormone were assessed. Thirty-four subjects completed the study. Weight loss occurred in ED1 (-3.8 ± 0.2 kg), ED2 (-2.8 ± 0.6 kg), and ED3 (-2.6 ± 1.1 kg) but was minimal in EXCON (-0.9 ± 0.7 kg). The overall sum of disturbances (luteal phase defects, anovulation, and oligomenorrhea) was greater in ED2 compared with EXCON and greater in ED3 compared with EXCON AND ED1. The average percent energy deficit was the main predictor of the frequency of menstrual disturbances (f = 10.1, ß = -0.48, r(2) = 0.23, P = 0.003) even when weight loss was included in the model. The estimates of the magnitude of energy deficiency associated with menstrual disturbances ranged from -22 (ED2) to -42% (ED3), reflecting an energy deficit of -470 to -810 kcal/day, respectively. This is the first study to demonstrate a dose-response relationship between the magnitude of energy deficiency and the frequency of exercise-related menstrual disturbances; however, the severity of menstrual disturbances was not dependent on the magnitude of energy deficiency.

Somatic reversion in dedicator of cytokinesis 8 immunodeficiency modulates disease phenotype.

BACKGROUND: Autosomal recessive loss-of-function mutations in dedicator of cytokinesis 8 (DOCK8) cause a combined immunodeficiency characterized by atopy, recurrent infections, and cancer susceptibility. A genotype-phenotype explanation for the variable disease expression is lacking. OBJECTIVE: We investigated whether reversions contributed to the variable disease expression. METHODS: Patients followed at the National Institutes of Health's Clinical Center were studied. We performed detailed genetic analyses and intracellular flow cytometry to detect DOCK8 protein expression within lymphocyte subsets. RESULTS: We identified 17 of 34 DOCK8-deficient patients who had germline mutations with variable degrees of reversion caused by somatic repair. Somatic repair of the DOCK8 mutations resulted from second-site mutation, original-site mutation, gene conversion, and intragenic crossover. Higher degrees of reversion were associated with recombination-mediated repair. DOCK8 expression was restored primarily within antigen-experienced T cells or natural killer cells but less so in naive T or B cells. Several patients exhibited multiple different repair events. Patients who had reversions were older and had less severe allergic disease, although infection susceptibility persisted. No patients were cured without hematopoietic cell transplantation. CONCLUSIONS: In patients with DOCK8 deficiency, only certain combinations of germline mutations supported secondary somatic repair. Those patients had an ameliorated disease course with longer survival but still had fatal complications or required hematopoietic cell transplantation. These observations support the concept that some DOCK8-immunodeficient patients have mutable mosaic genomes that can modulate disease phenotype over time.

Impaired T(H)17 cell differentiation in subjects with autosomal dominant hyper-IgE syndrome.

The autosomal dominant hyper-IgE syndrome (HIES, 'Job's syndrome') is characterized by recurrent and often severe pulmonary infections, pneumatoceles, eczema, staphylococcal abscesses, mucocutaneous candidiasis, and abnormalities of bone and connective tissue. Mutations presumed to underlie HIES have recently been identified in stat3, the gene encoding STAT3 (signal transducer and activator of transcription 3) (refs 3, 4). Although impaired production of interferon-gamma and tumour-necrosis factor by T cells, diminished memory T-cell populations, decreased delayed-type-hypersensitivity responses and decreased in vitro lymphoproliferation in response to specific antigens have variably been described, specific immunological abnormalities that can explain the unique susceptibility to particular infections seen in HIES have not yet been defined. Here we show that interleukin (IL)-17 production by T cells is absent in HIES individuals. We observed that ex vivo T cells from subjects with HIES failed to produce IL-17, but not IL-2, tumour-necrosis factor or interferon-gamma, on mitogenic stimulation with staphylococcal enterotoxin B or on antigenic stimulation with Candida albicans or streptokinase. Purified naive T cells were unable to differentiate into IL-17-producing (T(H)17) T helper cells in vitro and had lower expression of retinoid-related orphan receptor (ROR)-gammat, which is consistent with a crucial role for STAT3 signalling in the generation of T(H)17 cells. T(H)17 cells have emerged as an important subset of helper T cells that are believed to be critical in the clearance of fungal and extracellular bacterial infections. Thus, our data suggest that the inability to produce T(H)17 cells is a mechanism underlying the susceptibility to the recurrent infections commonly seen in HIES.

Extracellular Matrix Scaffold-Assisted Tumor Vaccines Induce Tumor Regression and Long-Term Immune Memory.

Injectable scaffold delivery is a strategy to enhance the efficacy of cancer vaccine immunotherapy. The choice of scaffold biomaterial is crucial, impacting both vaccine release kinetics and immune stimulation via the host response. Extracellular matrix (ECM) scaffolds prepared from decellularized tissues facilitate a pro-healing inflammatory response that promotes local cancer immune surveillance. Here, an ECM scaffold-assisted therapeutic cancer vaccine that maintains an immune microenvironment consistent with tissue reconstruction is engineered. Several immune-stimulating adjuvants are screened to develop a cancer vaccine formulated with decellularized small intestinal submucosa (SIS) ECM scaffold co-delivery. It is found that the STING pathway agonist cyclic di-AMP most effectively induces cytotoxic immunity in an ECM scaffold vaccine, without compromising key interleukin 4 (IL-4) mediated immune pathways associated with healing. ECM scaffold delivery enhances therapeutic vaccine efficacy, curing 50-75% of established E.G-7OVA lymphoma tumors in mice, while none are cured with soluble vaccine. SIS-ECM scaffold-assisted vaccination prolonged antigen exposure is dependent on CD8+ cytotoxic T cells and generates long-term antigen-specific immune memory for at least 10 months post-vaccination. This study shows that an ECM scaffold is a promising delivery vehicle to enhance cancer vaccine efficacy while being orthogonal to characteristics of pro-healing immune hallmarks.

Decreased luteinizing hormone pulse frequency is associated with elevated 24-hour ghrelin after calorie restriction and exercise in premenopausal women.

Elevated ghrelin has been shown to be associated with reduced luteinizing hormone (LH) pulsatility in Rhesus monkeys, rats, men, and recently women. We previously reported that 24-h ghrelin concentrations are elevated in women following a 3-mo exercise and diet program leading to weight loss. We investigated whether the elevations in ghrelin following an ~3-mo exercise and diet program leading to weight loss are associated with a decrease in LH pulsatility. The nonexercising control group (Control, n = 5) consumed a controlled diet that matched energy needs, whereas energy intake in the exercise group (Energy Deficit, n = 16) was reduced from baseline energy requirements and supervised exercise training occurred five times per a week. Significant decreases in body weight (-3.0 ± 0.6 kg), body fat (-2.9 ± 0.4 kg) and 24-h LH pulse frequency (-0.18 ± 0.08 pulses/h), and a significant increase in 24-h mean ghrelin were observed in only the Energy Deficit group. The pre-post change in LH pulse frequency was negatively correlated with the change in mean 24-h ghrelin (R = -0.485, P = 0.030) and the change in peak ghrelin at lunch (R = -0.518, P = 0.019). Interestingly, pre-post change in night LH pulse frequency was negatively correlated with the change in mean day ghrelin (R = -0.704, P = 0.001). Elevated total ghrelin concentrations are associated with the suppression of LH pulsatility in premenopausal women and may play a role in the suppression of reproductive function following weight loss.

Evolution of the donor T-cell repertoire in recipients in the second decade after allogeneic stem cell transplantation.

After allogeneic stem cell transplantation (SCT), T lymphocyte function is reestablished from the donor's postthymic T cells and through thymic T-cell neogenesis. The immune repertoire and its relation to that of the donor have not been characterized in detail in long-term adult SCT survivors. We studied 21 healthy patients in their second decade after a myeloablative SCT for hematologic malignancy (median follow-up, 12 years). Immune profiles were compared with donor samples cryopreserved at transplant and beyond 10 years from SCT. Only one recipient was on continuing immunosuppression. Compared with the donor at transplant, there was no significant difference in CD4, CD8, natural killer, and B-cell blood counts. However, compared with donors, recipients had significantly fewer naive T cells, lower T-cell receptor excision circle levels, fewer CD4 central memory cells, more effector CD8(+) cells, and more regulatory T cells. TCR repertoire analysis showed no significant difference in complexity of TCRVß spectratype between recipients and donors, although spectratype profiles had diverged with both gain and loss of donor repertoire peaks in the recipient. In conclusion, long-term allogeneic SCT survivors have subtle defects in their immune profile consistent with defective thymic function but compatible with normal health. This study is registered at http://www.clinicaltrials.gov as NCT00106925.

Hierarchical IL-5 expression defines a subpopulation of highly differentiated human Th2 cells.

Each of the three Th2 cytokine genes, IL-4, IL-5, and IL-13, has different functions. We hypothesized that Th2 heterogeneity could yield Th2 subpopulations with different cytokine expression and effector functions. Using multiple approaches, we demonstrate that human Th2 cells are composed of two major subpopulations: a minority IL-5(+) (IL-5(+), IL-4(+), IL-13(+)) and majority IL-5(-) Th2 (IL-5(-), IL-4(+), IL-13(+)) population. IL-5(+) Th2 cells comprised only 20% of all Th2 cells. Serial rounds of in vitro differentiation initially yielded IL-5(-) Th2, but required multiple rounds of differentiation to generate IL-5(+) Th2 cells. IL-5(+) Th2 cells expressed less CD27 and greater programmed cell death-1 than IL-5(-) Th2 cells, consistent with their being more highly differentiated, Ag-exposed memory cells. IL-5(+) Th2 cells expressed greater IL-4, IL-13, and GATA-3 relative to IL-5(-) Th2 cells. GATA-3 and H3K4me(3) binding to the IL5 promoter (IL5p) was greater in IL-5(+) relative to IL-5(-) Th2 cells, whereas there was no difference in their binding to the IL4p and IL13p. Conversely, H3K27me(3) binding to the IL5p was greater in IL-5(-) Th2 cells. These findings demonstrate Th2 lineage heterogeneity, in which the IL5 gene is regulated in a hierarchical manner relative to other Th2 genes. IL-5(+) Th2 cells are phenotypically distinct and have epigenetic changes consistent with greater IL5p accessibility. Recurrent antigenic exposure preferentially drives the differentiation of IL-5(+) Th2 cells. These results demonstrate that IL-5(+) and IL-5(-) Th2 cells, respectively, represent more and less highly differentiated Th2 cell subpopulations. Such Th2 subpopulations may differentially contribute to Th2-driven pathology.

Acquisition of direct antiviral effector functions by CMV-specific CD4+ T lymphocytes with cellular maturation.

The role of CD4+ T cells in the control of persistent viral infections beyond the provision of cognate help remains unclear. We used polychromatic flow cytometry to evaluate the production of the cytokines interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-2, the chemokine macrophage inflammatory protein (MIP)-1beta, and surface mobilization of the degranulation marker CD107a by CD4+ T cells in response to stimulation with cytomegalovirus (CMV)-specific major histocompatibility complex class II peptide epitopes. Surface expression of CD45RO, CD27, and CD57 on responding cells was used to classify CD4+ T cell maturation. The functional profile of virus-specific CD4+ T cells in chronic CMV infection was unique compared with that observed in other viral infections. Salient features of this profile were: (a) the simultaneous production of MIP-1beta, TNF-alpha, and IFN-gamma in the absence of IL-2; and (b) direct cytolytic activity associated with surface mobilization of CD107a and intracellular expression of perforin and granzymes. This polyfunctional profile was associated with a terminally differentiated phenotype that was not characterized by a distinct clonotypic composition. Thus, mature CMV-specific CD4+ T cells exhibit distinct functional properties reminiscent of antiviral CD8+ T lymphocytes.

A steady state of CD4+ T cell memory maturation and activation is established during primary subtype C HIV-1 infection.

The functional integrity of CD4(+) T cells is crucial for well-orchestrated immunity and control of HIV-1 infection, but their selective depletion during infection creates a paradox for understanding a protective response. We used multiparameter flow cytometry to measure activation, memory maturation, and multiple functions of total and Ag-specific CD4(+) T cells in 14 HIV-1- and CMV- coinfected individuals at 3 and 12 mo post HIV-1 infection. Primary HIV-1 infection was characterized by elevated levels of CD38, HLA-DR, and Ki67 in total memory and Gag-specific CD4(+) and CD8(+) T cells. In both HIV-infected and 15 uninfected controls, the frequency of activated cells was uniformly distributed among early differentiated (ED; CD45RO(+)CD27(+)), late differentiated (CD45RO(+)CD27(-)), and fully differentiated effector (CD45RO(-)CD27(-)) memory CD4(+) T cells. In HIV-1-infected individuals, activated CD4(+) T cells significantly correlated with viremia at 3 mo postinfection (r = 0.79, p = 0.0007) and also harbored more gag provirus DNA copies than nonactivated cells (p = 0.04). Moreover, Gag-specific ED CD4(+) T cells inversely associated with plasma viral load (r = -0.87, p < 0.0001). Overall, we show that low copy numbers of gag provirus and plasma RNA copies associated with low CD4 activation as well as accumulation of ED HIV-specific CD4(+) memory. Significant positive correlations between 3 and 12 mo activation and memory events highlighted that a steady state of CD4(+) T cell activation and memory maturation was established during primary infection and that these cells were unlikely to be involved in influencing the course of viremia in the first 12 mo of HIV-1 infection.

CCR2 identifies a stable population of human effector memory CD4+ T cells equipped for rapid recall response.

Because T cells act primarily through short-distance interactions, homing receptors can identify colocalizing cells that serve common functions. Expression patterns for multiple chemokine receptors on CD4(+) T cells from human blood suggested a hierarchy of receptors that are induced and accumulate during effector/memory cell differentiation. We characterized CD4(+)CD45RO(+) T cells based on expression of two of these receptors, CCR5 and CCR2, the principal subsets being CCR5(-)CCR2(-) (∼70%), CCR5(+)CCR2(-) (∼25%), and CCR5(+)CCR2(+) (∼5%). Relationships among expression of CCR5 and CCR2 and CD62L, and the subsets' proliferation histories, suggested a pathway of progressive effector/memory differentiation from the CCR5(-)CCR2(-) to CCR5(+)CCR2(-) to CCR5(+)CCR2(+) cells. Sensitivity and rapidity of TCR-mediated activation, TCR signaling, and effector cytokine production by the subsets were consistent with such a pathway. The subsets also showed increasing responsiveness to IL-7, and the CCR5(+)CCR2(+) cells were CD127(bright) and invariably showed the greatest response to tetanus toxoid. CCR5(+)CCR2(+) cells also expressed the largest repertoire of chemokine receptors and migrated to the greatest number of chemokines. By contrast, the CCR5(+)CCR2(-) cells had the greatest percentages of regulatory T cells, activated/cycling cells, and CMV-reactive cells, and were most susceptible to apoptosis. Our results indicate that increasing memory cell differentiation can be uncoupled from susceptibility to death, and is associated with an increase in chemokine responsiveness, suggesting that vaccination (or infection) can produce a stable population of effector-capable memory cells that are highly enriched in the CCR5(+)CCR2(+) subset and ideally equipped for rapid recall responses in tissue.

Lymphopenia and interleukin-2 therapy alter homeostasis of CD4+CD25+ regulatory T cells.

CD4(+)CD25(+) regulatory T (T(reg)) cells have a crucial role in maintaining immune tolerance. Mice and humans born lacking T(reg) cells develop severe autoimmune disease, and depletion of T(reg) cells in lymphopenic mice induces autoimmunity. Interleukin (IL)-2 signaling is required for thymic development, peripheral expansion and suppressive activity of T(reg) cells. Animals lacking IL-2 die of autoimmunity, which is prevented by administration of IL-2-responsive T(reg) cells. In light of the emerging evidence that one of the primary physiologic roles of IL-2 is to generate and maintain T(reg) cells, the question arises as to the effects of IL-2 therapy on them. We monitored T(reg) cells during immune reconstitution in individuals with cancer who did or did not receive IL-2 therapy. CD4(+)CD25(hi) cells underwent homeostatic peripheral expansion during immune reconstitution, and in lymphopenic individuals receiving IL-2, the T(reg) cell compartment was markedly increased. Mouse studies showed that IL-2 therapy induced expansion of existent T(reg) cells in normal hosts, and IL-2-induced T(reg) cell expansion was further augmented by lymphopenia. On a per-cell basis, T(reg) cells generated by IL-2 therapy expressed similar levels of FOXP3 and had similar potency for suppression compared to T(reg) cells present in normal hosts. These studies suggest that IL-2 and lymphopenia are primary modulators of CD4(+)CD25(+) T(reg) cell homeostasis.

Characterization of the diurnal rhythm of peptide YY and its association with energy balance parameters in normal-weight premenopausal women.

PYY may play a role in modulating satiety and energy expenditure; increasing PYY postprandially has been studied largely in single-meal responses. The diurnal rhythm of PYY and its role in energy balance have not been fully characterized. The purpose of our study was to characterize features of the diurnal rhythm of PYY and determine its role in regulating energy balance. This study was a cross-sectional analysis of 11 subjects in whom 24-h repeated blood sampling was conducted at baseline of a larger prospective study. Breakfast (B), lunch (L), dinner (D), and a snack (S) occurred between 0900 and 1900. Total PYY was assayed every hour from 0800 to 1000, every 20 min from 1000 to 2000, and every hour from 2000 to 0800. PYY variables included total AUC, postprandial peaks, and 24-h mean. Energy balance variables included energy intake, RMR, RQ, and NEAT. PYY postprandial peaks were significantly higher than fasting (P < 0.05). Twenty-four-hour peak PYY occurred after L and was significantly higher than all other peaks (P < 0.05). A cubic curve function accounted for most of the variance in PYY (r(2) = 69.9%, P < 0.01). Fasting PYY (0800) correlated with postprandial peaks at B (r = 0.77, P = 0.01), L (r = 0.71, P = 0.01), and D (r = 0.65, P = 0.03). The only significant association between PYY and energy expenditure was that RMR (kcal/24 h) correlated with 24-h mean PYY (r = 0.71, P = 0.013) and total AUC (r = 0.69, P = 0.019). We conclude that PYY displays a meal-driven diurnal rhythm and is correlated to RMR, a major contributor to energy expenditure. Thus, PYY varies in accordance with energy content and RMR, supporting a role for PYY in energy balance modulation.

Avidity for antigen shapes clonal dominance in CD8+ T cell populations specific for persistent DNA viruses.

The forces that govern clonal selection during the genesis and maintenance of specific T cell responses are complex, but amenable to decryption by interrogation of constituent clonotypes within the antigen-experienced T cell pools. Here, we used point-mutated peptide-major histocompatibility complex class I (pMHCI) antigens, unbiased TCRB gene usage analysis, and polychromatic flow cytometry to probe directly ex vivo the clonal architecture of antigen-specific CD8(+) T cell populations under conditions of persistent exposure to structurally stable virus-derived epitopes. During chronic infection with cytomegalovirus and Epstein-Barr virus, CD8(+) T cell responses to immunodominant viral antigens were oligoclonal, highly skewed, and exhibited diverse clonotypic configurations; TCRB CDR3 sequence analysis indicated positive selection at the protein level. Dominant clonotypes demonstrated high intrinsic antigen avidity, defined strictly as a physical parameter, and were preferentially driven toward terminal differentiation in phenotypically heterogeneous populations. In contrast, subdominant clonotypes were characterized by lower intrinsic avidities and proportionately greater dependency on the pMHCI-CD8 interaction for antigen uptake and functional sensitivity. These findings provide evidence that interclonal competition for antigen operates in human T cell populations, while preferential CD8 coreceptor compensation mitigates this process to maintain clonotypic diversity. Vaccine strategies that reconstruct these biological processes could generate T cell populations that mediate optimal delivery of antiviral effector function.

Long peptides induce polyfunctional T cells against conserved regions of HIV-1 with superior breadth to single-gene vaccines in macaques.

A novel T-cell vaccine strategy designed to deal with the enormity of HIV-1 variation is described and tested for the first time in macaques to inform and complement approaching clinical trials. T-cell immunogen HIVconsv, which directs vaccine-induced responses to the most conserved regions of the HIV-1, proteome and thus both targets diverse clades in the population and reduces the chance of escape in infected individuals, was delivered using six different vaccine modalities: plasmid DNA (D), attenuated human (A) and chimpanzee (C) adenoviruses, modified vaccinia virus Ankara (M), synthetic long peptides, and Semliki Forest virus replicons. We confirmed that the initial DDDAM regimen, which mimics one of the clinical schedules (DDDCM), is highly immunogenic in macaques. Furthermore, adjuvanted synthetic long peptides divided into sub-pools and delivered into anatomically separate sites induced T-cell responses that were markedly broader than those elicited by traditional single-open-reading-frame genetic vaccines and increased by 30% the overall response magnitude compared with DDDAM. Thus, by improving both the HIV-1-derived immunogen and vector regimen/delivery, this approach could induce stronger, broader, and theoretically more protective T-cell responses than vaccines previously used in humans.