A randomised controlled trial of long NY-ESO-1 peptide-pulsed autologous dendritic cells with or without alpha-galactosylceramide in high-risk melanoma.

AIM: We have previously reported that polyfunctional T cell responses can be induced to the cancer testis antigen NY-ESO-1 in melanoma patients injected with mature autologous monocyte-derived dendritic cells (DCs) loaded with long NY-ESO-1-derived peptides together with α-galactosylceramide (α-GalCer), an agonist for type 1 Natural Killer T (NKT) cells. OBJECTIVE: To assess whether inclusion of α-GalCer in autologous NY-ESO-1 long peptide-pulsed DC vaccines (DCV + α-GalCer) improves T cell responses when compared to peptide-pulsed DC vaccines without α-GalCer (DCV). DESIGN, SETTING AND PARTICIPANTS: Single-centre blinded randomised controlled trial in patients ≥ 18 years old with histologically confirmed, fully resected stage II-IV malignant cutaneous melanoma, conducted between July 2015 and June 2018 at the Wellington Blood and Cancer Centre of the Capital and Coast District Health Board. INTERVENTIONS: Stage I. Patients were randomised to two cycles of DCV or DCV + α-GalCer (intravenous dose of 10 × 106 cells, interval of 28 days). Stage II. Patients assigned to DCV + α-GalCer were randomised to two further cycles of DCV + α-GalCer or observation, while patients initially assigned to DCV crossed over to two cycles of DCV + α-GalCer. OUTCOME MEASURES: Primary: Area under the curve (AUC) of mean NY-ESO-1-specific T cell count detected by ex vivo IFN-γ ELISpot in pre- and post-treatment blood samples, compared between treatment arms at Stage I. Secondary: Proportion of responders in each arm at Stage I; NKT cell count in each arm at Stage I; serum cytokine levels at Stage I; adverse events Stage I; T cell count for DCV + α-GalCer versus observation at Stage II, T cell count before versus after cross-over. RESULTS: Thirty-eight patients gave written informed consent; 5 were excluded before randomisation due to progressive disease or incomplete leukapheresis, 17 were assigned to DCV, and 16 to DCV + α-GalCer. The vaccines were well tolerated and associated with increases in mean total T cell count, predominantly CD4+ T cells, but the difference between the treatment arms was not statistically significant (difference - 6.85, 95% confidence interval, - 21.65 to 7.92; P = 0.36). No significant improvements in T cell response were associated with DCV + α-GalCer with increased dosing, or in the cross-over. However, the NKT cell response to α-GalCer-loaded vaccines was limited compared to previous studies, with mean circulating NKT cell levels not significantly increased in the DCV + α-GalCer arm and no significant differences in cytokine response between the treatment arms. CONCLUSIONS: A high population coverage of NY-ESO-1-specific T cell responses was achieved with a good safety profile, but we failed to demonstrate that loading with α-GalCer provided an additional advantage to the T cell response with this cellular vaccine design. CLINICAL TRIAL REGISTRATION: ACTRN12612001101875. Funded by the Health Research Council of New Zealand.

pH and Proton Sensor GPR65 Determine Susceptibility to Atopic Dermatitis.

pH sensing by GPR65 regulates various inflammatory conditions, but its role in skin remains unknown. In this study, we performed a phenome-wide association study and report that the T allele of GPR65-intronic single-nucleotide polymorphism rs8005161, which reduces GPR65 signaling, showed a significant association with atopic dermatitis, in addition to inflammatory bowel diseases and asthma, as previously reported. Consistent with this genetic association in humans, we show that deficiency of GPR65 in mice resulted in markedly exacerbated disease in the MC903 experimental model of atopic dermatitis. Deficiency of GPR65 also increased neutrophil migration in vitro. Moreover, GPR65 deficiency in mice resulted in higher expression of the inflammatory cytokine TNF-α by T cells. In humans, CD4+ T cells from rs8005161 heterozygous individuals expressed higher levels of TNF-α after PMA/ionomycin stimulation, particularly under pH 6 conditions. pH sensing by GPR65 appears to be important for regulating the pathogenesis of atopic dermatitis.

Basophils promote barrier dysfunction and resolution in the atopic skin.

BACKGROUND: The type 2 cytokines IL-4 and IL-13 promote not only atopic dermatitis (AD) but also the resolution of inflammation. How type 2 cytokines participate in the resolution of AD is poorly known. OBJECTIVE: Our aim was to determine the mechanisms and cell types governing skin inflammation, barrier dysfunction, and resolution of inflammation in a model of AD. METHODS: Mice that exhibit expression of IL-4, IL-13, and MCPT8 or that could be depleted of basophils or eosinophils, be deficient in IL-4 or MHC class II molecules, or have basophils lacking macrophage colony-stimulating factor (M-CSF) were treated with calcipotriol (MC903) as an acute model of AD. Kinetics of the disease; keratinocyte differentiation; and leukocyte accumulation, phenotype, function, and cytokine production were measured by transepidermal water loss, histopathology, molecular biology, or unbiased analysis of spectral flow cytometry. RESULTS: In this model of AD, basophils were activated systemically and were the initial and main source of IL-4 in the skin. Basophils and IL-4 promoted epidermal hyperplasia and skin barrier dysfunction by acting on keratinocyte differentiation during inflammation. Basophils, IL-4, and basophil-derived M-CSF inhibited the accumulation of proinflammatory cells in the skin while promoting the expansion and function of proresolution M2-like macrophages and the expression of probarrier genes. Basophils kept their proresolution properties during AD resolution. CONCLUSION: Basophils can display both beneficial and detrimental type 2 functions simultaneously during atopic inflammation.

MR1-dependent immune surveillance of the skin contributes to pathogenesis and is a photobiological target of UV light therapy in a mouse model of atopic dermatitis.

BACKGROUND: Mucosal-associated invariant T (MAIT) cells are unconventional T cells which recognize microbial metabolites presented by the major histocompatibility complex class I-related molecule MR1. Although MAIT cells have been shown to reside in human and murine skin, their contribution to atopic dermatitis (AD), an inflammatory skin disease associated with barrier dysfunction and microbial translocation, has not yet been determined. METHODS: Genetic deletion of MR1 and topical treatment with inhibitory MR1 ligands, which result in the absence and functional inhibition of MAIT cells, respectively, were used to investigate the role of MR1-dependent immune surveillance in a MC903-driven murine model of AD. RESULTS: The absence or inhibition of MR1 arrested AD disease progression through the blockade of both eosinophil activation and recruitment of IL-4- and IL-13-producing cells. In addition, the therapeutic efficacy of phototherapy against MC903-driven AD could be increased with prior application of folate, which photodegrades into the inhibitory MR1 ligand 6-formylpterin. CONCLUSION: We identified MAIT cells as sentinels and mediators of cutaneous type 2 immunity. Their pathogenic activity can be inhibited by topical application or endogenous generation, via phototherapy, of inhibitory MR1 ligands.

Genetic regulation of antibody responsiveness to immunization in substrains of BALB/c mice.

Antibody-mediated immunity is highly protective against disease. The majority of current vaccines confer protection through humoral immunity, but there is high variability in responsiveness across populations. Identifying immune mechanisms that mediate low antibody responsiveness may provide potential strategies to boost vaccine efficacy. Here, we report diverse antibody responsiveness to unadjuvanted as well as adjuvanted immunization in substrains of BALB/c mice, resulting in high and low antibody response phenotypes. Furthermore, these antibody phenotypes were not affected by changes in environmental factors such as the gut microbiota composition. Antigen-specific B cells following immunization had a marked difference in capability to class switch, resulting in perturbed IgG isotype antibody production. In vitro, a B-cell intrinsic defect in the regulation of class-switch recombination was identified in mice with low IgG antibody production. Whole genome sequencing identified polymorphisms associated with the magnitude of antibody produced, and we propose candidate genes that may regulate isotype class-switching capability. This study highlights that mice sourced from different vendors can have significantly altered humoral immune response profiles, and provides a resource to interrogate genetic regulators of antibody responsiveness. Together these results further our understanding of immune heterogeneity and suggest additional research on the genetic influences of adjuvanted vaccine strategies is warranted for enhancing vaccine efficacy.

Enhancing T cell responses and tumour immunity by vaccination with peptides conjugated to a weak NKT cell agonist.

Activated NKT cells can stimulate antigen-presenting cells leading to enhanced peptide antigen-specific immunity. However, administration of potent NKT cell agonists like α-galactosylceramide (α-GalCer) can be associated with release of high levels of cytokines, and in some situations, hepatotoxicity. Here we show that it is possible to provoke sufficient NKT cell activity to stimulate strong antigen-specific T cell responses without these unwanted effects. This was achieved by chemically conjugating antigenic peptides to α-galactosylphytosphingosine (α-GalPhs), an NKT cell agonist with very weak activity based on structural characterisation and biological assays. Conjugation improved delivery to antigen-presenting cells in vivo, while use of a cathepsin-sensitive linker to release the α-GalPhs and peptide within the same cell promoted strong T cell activation and therapeutic anti-tumour responses in mice. The conjugates activated human NKT cells and enhanced human T cell responses to a viral peptide in vitro. Accordingly, we have demonstrated a means to safely exploit the immunostimulatory properties of NKT cells to enhance T cell activation for virus- and tumour-specific immunity.

Activation of Human Mucosal-Associated Invariant T Cells Induces CD40L-Dependent Maturation of Monocyte-Derived and Primary Dendritic Cells.

Mucosal-associated invariant T (MAIT) cells are innate T cells that recognize intermediates of the vitamin B2 biosynthetic pathway presented by the monomorphic MR1 molecule. It remains unclear whether, in addition to their cytolytic activity that is important in antimicrobial defense, MAIT cells have immune-modulatory functions that could enhance dendritic cell (DC) maturation. In this study, we investigated the molecular mechanisms dictating the interactions between human MAIT cells and DCs and demonstrate that human MAIT cells mature monocyte-derived and primary DCs in an MR1- and CD40L-dependent manner. Furthermore, we show that MAIT cell-derived signals synergize with microbial stimuli to induce secretion of bioactive IL-12 by DCs. Activation of human MAIT cells in whole blood leads to MR1- and cytokine-dependent NK cell transactivation. Our results underscore an important property of MAIT cells, which can be of translational relevance to rapidly orchestrate adaptive immunity through DC maturation.

A phase I vaccination study with dendritic cells loaded with NY-ESO-1 and α-galactosylceramide: induction of polyfunctional T cells in high-risk melanoma patients.

Vaccines that elicit targeted tumor antigen-specific T-cell responses have the potential to be used as adjuvant therapy in patients with high risk of relapse. However, the responses induced by vaccines in cancer patients have generally been disappointing. To improve vaccine function, we investigated the possibility of exploiting the immunostimulatory capacity of type 1 Natural killer T (NKT) cells, a cell type enriched in lymphoid tissues that can trigger improved antigen-presenting function in dendritic cells (DCs). In this phase I dose escalation study, we treated eight patients with high-risk surgically resected stage II-IV melanoma with intravenous autologous monocyte-derived DCs loaded with the NKT cell agonist α-GalCer and peptides derived from the cancer testis antigen NY-ESO-1. Two synthetic long peptides spanning defined immunogenic regions of the NY-ESO-1 sequence were used. This therapy proved to be safe and immunologically effective, inducing increases in circulating NY-ESO-1-specific T cells that could be detected directly ex vivo in seven out of eight patients. These responses were achieved using as few as 5 × 105 peptide-loaded cells per dose. Analysis after in vitro restimulation showed increases in polyfunctional CD4+ and CD8+ T cells that were capable of manufacturing two or more cytokines simultaneously. Evidence of NKT cell proliferation and/or NKT cell-associated cytokine secretion was seen in most patients. In light of these strong responses, the concept of including NKT cell agonists in vaccine design requires further investigation.

A self-adjuvanting vaccine induces cytotoxic T lymphocytes that suppress allergy.

Epitope-based peptide vaccines encompass minimal immunogenic regions of protein antigens to allow stimulation of precisely targeted adaptive immune responses. However, because efficacy is largely determined by the functional status of antigen-presenting cells (APCs) that acquire and present peptides to cells of the adaptive immune system, adjuvant compounds are needed to enhance immunogenicity. We present here a vaccine consisting of an allergen-derived peptide conjugated to a prodrug of the natural killer-like T (NKT) cell agonist α-galactosylceramide, which is highly effective in reducing inflammation in a mouse model of allergic airway inflammation. Unlike other peptide-adjuvant conjugates that directly activate APCs through pattern recognition pathways, this vaccine encourages third-party interactions with NKT cells to enhance APC function. Therapeutic efficacy was correlated with marked increases in the number and functional activity of allergen-specific cytotoxic T lymphocytes (CTLs), leading to suppression of immune infiltration into the lungs after allergen challenge in sensitized hosts.

Dendritic cell vaccination combined with temozolomide retreatment: results of a phase I trial in patients with recurrent glioblastoma multiforme.

There is no standard treatment for recurrent glioblastoma multiforme (GBM). Retreatment with temozolomide (TMZ) is one treatment option. We reasoned this could be more effective if combined with a vaccine that preferentially targeted TMZ-resistant cells. To test the feasibility and safety of such an approach, a phase 1 trial was conducted in which patients with GBM tumors relapsing after standard chemoradiotherapy were retreated with TMZ in combination with a vaccine consisting of monocyte-derived dendritic cells (DC) pulsed with autologous tumor cells that had previously been exposed to TMZ in vivo in the course of primary treatment. Of 14 participants, nine patients completed the initial phase of priming vaccinations and two cycles of TMZ, one proved to have radionecrosis, one rapidly progressed, and in three the yield of DC vaccine was insufficient to proceed with treatment. Other than expected toxicities related to TMZ, there were no adverse events attributable to the combined treatment. Two patients had objective radiological responses. Six month progression-free survival was 22 %, similar to retreatment with TMZ alone. Anti-tumor immune responses were assessed in peripheral blood mononuclear cells using interferon-γ ELISpot, with two patients meeting criteria for a vaccine-induced immune response, one of whom remained disease-free for nearly three years. Another patient with an anti-tumor immune response at baseline that was sustained post-vaccination experienced a 12-month period of progression-free survival. In summary, the combined treatment was safe and well-tolerated but feasibility in the recurrent setting was marginal. Evidence of immune responses in a few patients broadly correlated with better clinical outcome.

Epstein-Barr virus negativity among individuals older than 60 years is associated with HLA-C and HLA-Bw4 variants and tonsillectomy.

Epstein-Barr virus (EBV) infects ≈ 95% of the adult population. The factors that confer protection in the remaining ≈ 5% remain unknown. In an exploratory study, we assessed immunogenetic factors and tonsillectomy in a cohort of 17 EBV-negative and 39 EBV-positive healthy individuals aged >60 years. Analyses of HLA genotypes revealed an association between EBV negativity and the presence of HLA-C-35T/T and/or HLA-Bw4 alleles. In addition, EBV-negative donors presented with a history of tonsillectomy more often than EBV-positive donors.

Whole genome deep sequencing of HIV-1 reveals the impact of early minor variants upon immune recognition during acute infection.

Deep sequencing technologies have the potential to transform the study of highly variable viral pathogens by providing a rapid and cost-effective approach to sensitively characterize rapidly evolving viral quasispecies. Here, we report on a high-throughput whole HIV-1 genome deep sequencing platform that combines 454 pyrosequencing with novel assembly and variant detection algorithms. In one subject we combined these genetic data with detailed immunological analyses to comprehensively evaluate viral evolution and immune escape during the acute phase of HIV-1 infection. The majority of early, low frequency mutations represented viral adaptation to host CD8+ T cell responses, evidence of strong immune selection pressure occurring during the early decline from peak viremia. CD8+ T cell responses capable of recognizing these low frequency escape variants coincided with the selection and evolution of more effective secondary HLA-anchor escape mutations. Frequent, and in some cases rapid, reversion of transmitted mutations was also observed across the viral genome. When located within restricted CD8 epitopes these low frequency reverting mutations were sufficient to prime de novo responses to these epitopes, again illustrating the capacity of the immune response to recognize and respond to low frequency variants. More importantly, rapid viral escape from the most immunodominant CD8+ T cell responses coincided with plateauing of the initial viral load decline in this subject, suggestive of a potential link between maintenance of effective, dominant CD8 responses and the degree of early viremia reduction. We conclude that the early control of HIV-1 replication by immunodominant CD8+ T cell responses may be substantially influenced by rapid, low frequency viral adaptations not detected by conventional sequencing approaches, which warrants further investigation. These data support the critical need for vaccine-induced CD8+ T cell responses to target more highly constrained regions of the virus in order to ensure the maintenance of immunodominant CD8 responses and the sustained decline of early viremia.

Polyphenol and glucosinolate-derived AhR modulators regulate GPR15 expression on human CD4+ T cells.

Diets high in fruit and vegetables are perceived to be beneficial for intestinal homeostasis, in health as well as in the context of inflammatory bowel diseases (IBDs). Recent breakthroughs in the field of immunology have highlighted the importance of the ligand-activated transcription factor aryl hydrocarbon receptor (AhR) as a critical regulator of mucosal immunity, including the intestinal trafficking of CD4+ helper T cells, an immune cell subset implicated in a wide range of homeostatic and pathogenic processes. Specifically, the AhR has been shown to directly regulate the expression of the chemoattractant receptor G Protein-Coupled Receptor 15 (GPR15) on CD4+ T cells. GPR15 is an important gut homing marker whose expression on CD4+ T cells in the peripheral circulation is elevated in patients suffering from ulcerative colitis, raising the possibility that, in this setting, the beneficial effect of a diet rich in fruits and vegetables may be mediated through the modulation of GPR15 expression. To address this, we screened physiologically-relevant polyphenol and glucosinolate metabolites for their ability to affect both AhR activity and GPR15 expression. Our complementary approach and associated findings suggest that polyphenol and glucosinolate metabolites can regulate GPR15 expression on human CD4+ T cells in an AhR-dependent manner.

Controlled Hookworm Infection for Medication-free Maintenance in Patients with Ulcerative Colitis: A Pilot, Double-blind, Randomized Control Trial.

BACKGROUND: Human hookworm has been proposed as a treatment for ulcerative colitis (UC). This pilot study assessed the feasibility of a full-scale randomized control trial examining hookworm to maintain clinical remission in patients with UC. METHODS: Twenty patients with UC in disease remission (Simple Clinical Colitis Activity Index [SCCAI] ≤4 and fecal calprotectin (fCal) <100 ug/g) and only on 5-aminosalicylate received 30 hookworm larvae or placebo. Participants stopped 5-aminosalicylate after 12 weeks. Participants were monitored for up to 52 weeks and exited the study if they had a UC flare (SCCAI ≥5 and fCal ≥200 µg/g). The primary outcome was difference in rates of clinical remission at week 52. Differences were assessed for quality of life (QoL) and feasibility aspects including recruitment, safety, effectiveness of blinding, and viability of the hookworm infection. RESULTS: At 52 weeks, 4 of 10 (40%) participants in the hookworm group and 5 of 10 (50%) participants in the placebo group had maintained clinical remission (odds ratio, 0.67; 95% CI, 0.11-3.92). Median time to flare in the hookworm group was 231 days (interquartile range [IQR], 98-365) and 259 days for placebo (IQR, 132-365). Blinding was quite successful in the placebo group (Bang's blinding index 0.22; 95% CI, -0.21 to 1) but less successful in the hookworm group (0.70; 95% CI, 0.37-1.0). Almost all participants in the hookworm group had detectable eggs in their faeces (90%; 95% CI, 0.60-0.98), and all participants in this group developed eosinophilia (peak eosinophilia 4.35 × 10^9/L; IQR, 2.80-6.68). Adverse events experienced were generally mild, and there was no significant difference in QoL. CONCLUSIONS: A full-scale randomized control trial examining hookworm therapy as a maintenance treatment in patients with UC appears feasible.

This pilot study has shown a full-scale RCT examining hookworm therapy as maintenance therapy in patients with ulcerative colitis is feasible, safe, and will be well-tolerated.

Antigen-specific adaptive immune responses in fingolimod-treated multiple sclerosis patients.

T cells exit secondary lymphoid organs along a sphingosine1-phosphate (S1P) gradient and, accordingly, are reduced in blood upon fingolimod-mediated S1P-receptor (S1PR)-blockade. Serving as a model of adaptive immunity, we characterized cellular and humoral immune responses to influenza vaccine in fingolimod-treated patients with multiple sclerosis (MS) and in untreated healthy controls. Although the mode of action of fingolimod might predict reduced immunity, vaccine-triggered T cells accumulated normally in blood despite efficient S1PR-blockade. Concentrations of anti-influenza A/B immunoglobulin (Ig)M and IgG also increased similarly in both groups. These results indicate that fingolimod-treated individuals can mount vaccine-specific adaptive immune responses comparable to healthy controls.

Importance of the endometrial immune environment in endometrial cancer and associated therapies.

Endometrial cancer is rising in prevalence. The standard treatment modality of hysterectomy is becoming increasingly inadequate due primarily to the direct link between endometrial cancer and high BMI which increases surgical risks. This is an immunogenic cancer, with unique molecular subtypes associated with differential immune infiltration. Despite the immunogenicity of endometrial cancer, there is limited pre-clinical and clinical evidence of the function of immune cells in both the normal and cancerous endometrium. Immune checkpoint inhibitors for endometrial cancer are the most well studied type of immune therapy but these are not currently used as standard-of-care and importantly, they represent only one method of immune manipulation. There is limited evidence regarding the use of other immunotherapies as surgical adjuvants or alternatives. Levonorgestrel-loaded intra-uterine systems can also be effective for early-stage disease, but with varying success. There is currently no known reason as to what predisposes some patients to respond while others do not. As hormones can directly influence immune cell function, it is worth investigating the immune compartment in this context. This review assesses the immunological components of the endometrium and describes how the immune microenvironment changes with hormones, obesity, and in progression to malignancy. It also describes the importance of investigating novel pathways for immunotherapy.

MR1-dependence of unmetabolized folic acid side-effects.

The fortification of flour with folic acid for the prevention of neural tube defects (NTD) is currently mandated in over eighty countries worldwide, hence compelling its consumption by the greater part of the world's population. Notwithstanding its beneficial impact on rates of NTD, pervasive folic acid supplementation has invariably led to additive daily intakes reaching well beyond their original target, resulting in the circulation of unmetabolized folic acid. Associated idiopathic side-effects ranging from allergies to cancer have been suggested, albeit inconclusively. Herein, we hypothesize that their inconsistent detection and elusive etiology are linked to the in vivo generation of the immunosuppressive folic acid metabolite 6-formylpterin, which interferes with the still emerging and varied functions of Major Histocompatibility Complex-related molecule 1 (MR1)-restricted T cells. Accordingly, we predict that fortification-related adverse health outcomes can be eliminated by substituting folic acid with the bioequivalent folate vitamer 5-methyltetrahydrofolate, which does not break down into 6-formylpterin.

"Nourish to Flourish": complementary feeding for a healthy infant gut microbiome-a non-randomised pilot feasibility study.

BACKGROUND: The introduction of complementary foods and changes in milk feeding result in modifications to gastrointestinal function. The interplay between indigestible carbohydrates, host physiology, and microbiome, and immune system development are areas of intense research relevant to early and later-life health. METHODS: This 6-month prospective non-randomised feasibility study was conducted in Auckland, New Zealand (NZ), in January 2018. Forty parents/caregivers and their infants were enrolled, with 30 infants allocated to receive a prebiotic NZ kumara (flesh and skin; a type of sweet potato) prepared as a freeze-dried powder, and ten infants allocated to receive a commercially available probiotic control known to show relevant immune benefits (109 CFU Bifidobacterium lactis BB-12®). The primary outcome was the study feasibility measures which are reported here. RESULTS: Recruitment, participant retention, and data collection met feasibility targets. Some limitations to biological sample collection were encountered, with difficulties in obtaining sufficient plasma sample volumes for the proposed immune parameter analyses. Acceptability of the kumara powder was met with no reported adverse events. CONCLUSION: This study indicates that recruiting infants before introducing complementary foods is feasible, with acceptable adherence to the food-based intervention. These results will inform the protocol of a full-scale randomised controlled trial (RCT) with adjustments to the collection of biological samples to examine the effect of a prebiotic food on the prevalence of respiratory tract infections during infancy. Trial registration Australia New Zealand Clinical Trials Registry ACTRN12618000157279 . Prospectively registered on 02/01/2018.

A high-mobility, low-cost phenotype defines human effector-memory CD8+ T cells.

T cells move randomly ("random-walk"), a characteristic thought to be integral to their function. Using migration assays and time-lapse microscopy, we found that CD8+ T cells lacking the lymph node homing receptors CCR7 and CD62L migrate more efficiently in transwell assays, and that these same cells are characterized by a high frequency of cells exhibiting random crawling activity under culture conditions mimicking the interstitial/extravascular milieu, but not when examined on endothelial cells. To assess the energy efficiency of cells crawling at a high frequency, we measured mRNA expression of genes key to mitochondrial energy metabolism (peroxisome proliferator-activated receptor gamma coactivator 1beta [PGC-1beta], estrogen-related receptor alpha [ERRalpha], cytochrome C, ATP synthase, and the uncoupling proteins [UCPs] UCP-2 and -3), quantified ATP contents, and performed calorimetric analyses. Together these assays indicated a high energy efficiency of the high crawling frequency CD8+ T-cell population, and identified differentially regulated heat production among nonlymphoid versus lymphoid homing CD8+ T cells.