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Infecting humans with controlled doses of small intestinal helminths, such as human hookworm, is proposed as a therapy for the colonic inflammatory disease ulcerative colitis. Strengthening the colonic mucus barrier is a potential mechanism by which small intestinal helminths could treat ulcerative colitis. In this study, we compare C57BL/6 mice infected with the small intestinal helminth Heligmosomoides polygyrus and uninfected controls to investigate changes in colonic mucus. Histology, gene expression, and immunofluorescent analysis demonstrate that this helminth induces goblet cell hyperplasia, and an upregulation of mucin sialylation, and goblet-cell-derived functional proteins resistin-like molecule-beta (RELM-β) and trefoil factors (TFFs), in the colon. Using IL-13 knockout mice, we reveal that these changes are predominantly IL-13-dependent. The assessment of the colonic mucus microbiome demonstrates that H. polygyrus infection increases the abundance of Ruminococcus gnavus , a commensal bacterium capable of utilising sialic acid as an energy source. This study also investigates a human cohort experimentally challenged with human hookworm. It demonstrates that TFF blood levels increase in individuals chronically infected with small intestinal helminths, highlighting a conserved mucus response between humans and mice. Overall, small intestinal helminths modify colonic mucus, highlighting this as a plausible mechanism by which human hookworm therapy could treat ulcerative colitis.

Background: The introduction of complementary foods during the first year of life influences the diversity of the gut microbiome. How this diversity affects immune development and health is unclear., Objective: This study evaluates the effect of consuming kūmara or kūmara with added banana powder (resistant starch) compared to a reference control at 4 months post randomization on the prevalence of respiratory tract infections and the development of the gut microbiome., Methods: This study is a double-blind, randomized controlled trial of mothers and their 6-month-old infants (up to n=300) who have not yet started solids. Infants are randomized into one of 3 groups: control arm (C), standard kūmara intervention (K), and a kūmara intervention with added banana powder product (K+) to be consumed daily for 4 months until the infant is approximately 10 months old. Infants are matched for sex using stratified randomization. Data are collected at baseline (prior to commencing solid food) and at 2 and 4 months after commencing solid food (at around 8 and 10 months of age). Data and samples collected at each timepoint include weight and length, intervention adherence (months 2 and 4), illness and medication history, dietary intake (months 2 and 4), sleep (diary and actigraphy), maternal dietary intake, breast milk, feces (baseline and 4 months), and blood samples (baseline and 4 months)., Results: The trial was approved by the Health and Disability Ethics Committee of the Ministry of Health, New Zealand (reference 20/NTA/9). Recruitment and data collection did not commence until January 2022 due to the COVID-19 pandemic. Data collection and analyses are expected to conclude in January 2024 and early 2025, respectively. Results are to be published in 2024 and 2025., Conclusions: The results of this study will help us understand how the introduction of a specific prebiotic complementary food affects the microbiota and relative abundances of the microbial species, the modulation of immune development, and infant health. It will contribute to the expanding body of research that aims to deepen our understanding of the connections between nutrition, gut microbiota, and early-life postnatal health., Trial Registration: Australian New Zealand Clinical Trials Registry ACTRN12620000026921; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=378654., International Registered Report Identifier (irrid): DERR1-10.2196/56772., (©Clare R Wall, Nicole C Roy, Jane A Mullaney, Warren Charles McNabb, Olivier Gasser, Karl Fraser, Eric Altermann, Wayne Young, Janine Cooney, Robyn Lawrence, Yannan Jiang, Barbara C Galland, Xiaoxi Fu, Jacqueline N Tonkie, Nisha Mahawar, Amy Luisa Lovell. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 02.09.2024.)

Infecting humans with controlled doses of helminths, such as human hookworm (termed hookworm therapy), is proposed to prevent or treat various intestinal and extraintestinal diseases. However, full-scale clinical trials examining hookworm therapy are limited by the inability to scale-up the production of hookworm larvae to infect sufficient numbers of patients. With the aim of overcoming this challenge, this study infected four healthy individuals with hookworm larvae that had been reanimated from cryopreserved eggs to examine their viability and immunogenicity. We demonstrate that reanimated cryopreserved hookworm larvae establish a viable hookworm infection and elicit a similar immune response to larvae cultured from fresh stool. Furthermore, a refined understanding of the therapeutic mechanisms of hookworm is imperative to determine which diseases to target with hookworm therapy. To investigate potential therapeutic mechanisms, this study assessed changes in the immune cells, microbiome, and plasma metabolome in the four healthy individuals infected with cryopreserved hookworm larvae and another nine individuals infected with larvae cultured from freshly obtained stool. We identified potential immunoregulatory mechanisms by which hookworm may provide a beneficial effect on its host, including increased expression of CTLA-4 on regulatory T cells (Tregs) and upregulation of tryptophan metabolism. Furthermore, we found that a participant's baseline microbiome predicted the severity of symptoms and intestinal inflammation experienced during a controlled hookworm infection. In summary, our findings demonstrate the feasibility of full-scale clinical trials examining hookworm therapy by minimizing the reliance on human donors and optimizing the culturing process, thereby enabling viable hookworm larvae to be mass-produced and enabling on-demand inoculation of patients. Furthermore, this study provides insights into the complex interactions between helminths and their host, which could inform the development of novel therapeutic strategies.

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 × 10 6 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., (© 2023. The Author(s).)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 van der Woude, Hally, Currie, Gasser and Henry.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tang, Cait, White, Arabshahi, O’Sullivan and Gasser.)

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 kūmara (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 (10 9 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 kūmara 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., (© 2022. The Author(s).)

Influenza vaccination is an effective public health measure to reduce the risk of influenza illness, particularly when the vaccine is well matched to circulating strains. Notwithstanding, the efficacy of influenza vaccination varies greatly among vaccinees due to largely unknown immunological determinants, thereby dampening population-wide protection. Here, we report that dietary fibre may play a significant role in humoral vaccine responses. We found dietary fibre intake and the abundance of fibre-fermenting intestinal bacteria to be positively correlated with humoral influenza vaccine-specific immune responses in human vaccinees, albeit without reaching statistical significance. Importantly, this correlation was largely driven by first-time vaccinees; prior influenza vaccination negatively correlated with vaccine immunogenicity. In support of these observations, dietary fibre consumption significantly enhanced humoral influenza vaccine responses in mice, where the effect was mechanistically linked to short-chain fatty acids, the bacterial fermentation product of dietary fibre. Overall, these findings may bear significant importance for emerging infectious agents, such as COVID-19, and associated de novo vaccinations., Competing Interests: SE and GW were employed by Food Savvy. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest., (Copyright © 2021 Cait, Mooney, Poyntz, Shortt, Jones, Gestin, Gell, Grooby, O’Sullivan, Tang, Young, Thayabaran, Sparks, Ostapowicz, Tay, Poppitt, Elliott, Wakefield, Parry-Strong, Ralston, Beasley, Weatherall, Braithwaite, Forbes-Blom and Gasser.)

The immune system plays a significant role in controlling systemic metabolism. Innate-like T (ILT) cells in particular, such as mucosal-associated invariant T (MAIT) cells, invariant natural killer T (iNKT) cells and γδ T cell receptor expressing cells, have been reported to promote metabolic homeostasis. However, these different ILT cell subsets have, to date, been generally studied in isolation. Here we conducted a pilot study assessing the phenotype and function of circulating MAIT, iNKT, and Vδ2 + T cells in a small cohort of 10 people with obesity and type 2 diabetes (T2D), 10 people with obesity but no diabetes, and 12 healthy individuals. We conducted phenotypic analysis by flow cytometry ex vivo , and then functional analysis after in vitro stimulation using either PMA/ionomycin or synthetic agonists, or precursors thereof, for each of the cell-types; use of the latter may provide important knowledge for the development of novel therapeutics aimed at activating human ILT cells. The results of our pilot study, conducted on circulating cells, show clear dysfunction of all three ILT cell subsets in obese and obese T2D patients, as compared to healthy controls. Importantly, while both iNKT and Vδ2 + T cell dysfunctions were characterized by diminished IL-2 and interferon-γ production, the distinct dysfunctional state of MAIT cells was instead defined by skewed subset composition, heightened sensitivity to T cell receptor engagement and unchanged production of all measured cytokines., (Copyright © 2020 Li, Woods, Parry-Strong, Anderson, Capistrano, Gestin, Painter, Hermans, Krebs and Gasser.)

Background: Weaning is a period of marked physiological change. The introduction of solid foods and the changes in milk consumption are accompanied by significant gastrointestinal, immune, developmental, and microbial adaptations. Defining a reduced number of infections as the desired health benefit for infants around weaning, we identified in silico (i.e., by advanced public domain mining) infant gut microbes as potential deliverers of this benefit. We then investigated the requirements of these bacteria for exogenous metabolites as potential prebiotic feeds that were subsequently searched for in the natural product space., Results: Using public domain literature mining and an in silico reverse metabolic approach, we constructed probiotic-prebiotic-food associations, which can guide targeted feeding of immune health-beneficial microbes by weaning food; analyzed competition and synergy for (prebiotic) nutrients between selected microbes; and translated this information into designing an experimental complementary feed for infants enrolled in a pilot clinical trial ( http://www.nourishtoflourish.auckland.ac.nz/ )., Conclusions: In this study, we applied a benefit-oriented microbiome research strategy for enhanced early-life immune health. We extended from "classical" to molecular nutrition aiming to identify nutrients, bacteria, and mechanisms that point towards targeted feeding to improve immune health in infants around weaning. Here, we present the systems biology-based approach we used to inform us on the most promising prebiotic combinations known to support growth of beneficial gut bacteria ("probiotics") in the infant gut, thereby favorably promoting development of the immune system.

Objective: We investigated the potential feasibility of a randomized controlled trial of a nutritional intervention that may alter human gut microbiota and support immune defence against respiratory tract infection in adults (Proposed Study)., Methods: In total, 125 healthy adults aged 18-64 participated in a 6-month study that measured antibody response to the seasonal trivalent influenza vaccine. We assessed completion rates, procedure adherence rates and the influence of possible exclusion criteria on potential recruitment into the Proposed Study. We examined whether the gut microbiota could be categorised into enterotypes, and whether there was an association between enterotypes and the antibody response to the influenza vaccine., Results: The participant completion rate was 97.6% (95% CI 93.1-99.5%). The proportions (95% CI) of participants who may be excluded for antibiotic or corticosteroid use in the 30 days prior to the study, or due to receiving the influenza vaccine in the previous two years were 9.6% (5.1-16.2), 8.0% (3.9-14.2) and 61.6% (52.5-70.2), respectively. All participants were stratified into four gut microbiota enterotypes. There was no association between these enterotypes and the antibody response to the influenza vaccine, although the study was not powered for this outcome., Conclusion: This study design is suitable for the Proposed Study. The completion rate is likely to be high, although exclusion criteria should be selected with care. Further analyses of gut microbiota composition or function in association with antibody and immune responses are warranted to explore the role of host-microbiota interactions on protective immunity.

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 × 10 5 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.

Ex vivo generated monocyte-derived dendritic cell (moDC)-vaccines have long been touted as promising immunotherapeutic agents for cancer treatment, although the response rate generally remains low. The reasons for this are still unclear and confounded by the diversity in manufacturing protocols that may affect moDC function. Preclinical studies have shown that the stimulatory function of dendritic cells can be improved by engaging invariant NKT cells in vivo through the presentation of the glycolipid alpha-galactosylceramide via CD1d. However, expression of CD1d on moDC has been shown to be negatively correlated with expression of CD1a, which in turn has been suggested to be a surrogate marker for IL-12 secreting type-1 polarized moDC, the preferred functional characteristics for cancer vaccines. Here we challenge this notion by showing that plasma-derived lipids drive functional levels of CD1d expression, while CD1a expression can vary considerably in these cells without being correlated with a loss of polarization or immunogenicity.

It is known that T cells can eliminate tumour cells through recognition of unique or aberrantly expressed antigens presented as peptide epitopes by major histocompatibility complex (MHC) molecules on the tumour cell surface. With recent advances in defining tumour-associated antigens, it should now be possible to devise therapeutic vaccines that expand specific populations of anti-tumour T cells. However there remains a need to develop simpler efficacious synthetic vaccines that possess clinical utility. We present here the synthesis and analysis of vaccines based on conjugation of MHC-binding peptide epitopes to α-galactosylceramide, a glycolipid presented by the nonpolymorphic antigen-presenting molecule CD1d to provoke the stimulatory activity of type I natural killer T (NKT) cells. The chemical design incorporates an enzymatically cleavable linker that effects controlled release of the active components in vivo . Chemical and biological analysis of different linkages with different enzymatic targets enabled selection of a synthetic vaccine construct with potent therapeutic anti-tumour activity in mice, and marked in vitro activity in human blood.

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.

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.

Unlabelled: A key limitation in developing radiotherapeutic proteins is the expense of manufacturing the drug in small batches using traditional reaction vessels. Removing limitations on the quantity of protein labeled at any one time significantly decreases the cost of production, and nowhere is the need for cost-effective radiotherapeutics more acute than in the treatment of cancer., Methods: We describe a novel method that can rapidly radiolabel, theoretically, unlimited amounts of protein, without causing significant damage to binding potency or structural integrity. Our process controls the reaction rate for the isotope and reactants as they simultaneously flow through a reaction tube., Results: We have demonstrated proof of principle by labeling nearly a gram of antibody with 481 GBq (13 Ci) of (131)I during a single 30-min reaction run., Conclusion: Simple to construct, our system is already used to manufacture a radiolabeled antibody, both in the United States and in India, as part of clinical trials to treat glioblastoma multiforme. Modified, this system may be also applicable for nonradioactive labeling.

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.

Background: In chronic HIV infection, antiretroviral therapy-induced normalization of CD4(+) T cell counts (immune reconstitution [IR]) is associated with a decreased incidence of opportunistic diseases. However, some individuals remain at risk for opportunistic diseases despite prolonged normalization of CD4(+) T cell counts. Deficient Epstein-Barr virus (EBV)-specific CD4(+) T cell function may explain the occurrence of EBV-associated opportunistic malignancy-such as primary central nervous system (PCNS) lymphoma-despite recovery of absolute CD4(+) T cell counts., Methods and Findings: Absolute CD4(+) T cell counts and EBV-specific CD4(+) T cell-dependent interferon-gamma production were assessed in six HIV-positive individuals prior to development of PCNS lymphoma ("cases"), and these values were compared with those in 16 HIV-infected matched participants with no sign of EBV-associated pathology ("matched controls") and 11 nonmatched HIV-negative blood donors. Half of the PCNS lymphoma patients fulfilled IR criteria (defined here as CD4(+) T cell counts >or=500/microl blood). EBV-specific CD4(+) T cells were assessed 0.5-4.7 y prior to diagnosis of lymphoma. In 0/6 cases versus 13/16 matched controls an EBV-specific CD4(+) T cell response was detected (p = 0.007; confidence interval for odds ratio [0-0.40]). PCNS lymphoma patients also differed with regards to this response significantly from HIV-negative blood donors (p < 0.001, confidence interval for odds ratio [0-0.14]), but there was no evidence for a difference between HIV-negative participants and the HIV-positive matched controls (p = 0.47)., Conclusions: Irrespective of absolute CD4(+) T cell counts, HIV-positive patients who subsequently developed PCNS lymphoma lacked EBV-specific CD4(+) T cell function. Larger, ideally prospective studies are needed to confirm these preliminary data, and clarify the impact of pathogen-specific versus surrogate marker-based assessment of IR on clinical outcome.

Activation and subsequent differentiation of naive CD8+ T cells lead to the development of memory subsets with distinct homing and effector capacities. On nonlymphoid homing subsets, expression of "inflammatory" chemokine receptors (such as CXCR3, CCR5, CX3CR1, and CXCR1) is believed to promote migration into sites of infection/inflammation. Here we show that CXCR1 can be up-regulated to the cell surface within minutes of activating human CD8+ T cells. No concurrent up-regulation of other inflammatory chemokine receptors was observed. Up-regulation of CXCR1 preferentially occurred on central memory CD8+ T cells-that is, cells with a lymph node homing phenotype-and was functionally relevant. Immunofluorescence microscopy showed CXCR1 to be present in intracellular vesicles that do not significantly colocalize with perforin, RANTES (regulated upon activation normal T cell expressed and secreted), or the lysosomal marker CD63. By contrast, partial colocalization with the Golgi marker GM130, the constitutive secretory pathway marker beta2-microglobulin, and the early endosome marker EEA1 was observed. Up-regulation of CXCR1 did not occur after T-cell receptor cross-linking. By contrast, supernatants from activated neutrophils, but not from monocytes or dendritic cells, induced its up-regulation. These results suggest that CD8+ T cells can rapidly adapt their homing properties by mobilizing CXCR1 from a distinct intracellular compartment.

On activation, human neutrophils release microparticles, called ectosomes, directly from the cell surface membrane. Microparticles from platelets, endothelial cells, and monocytes were reported to support coagulation or to modulate vascular homeostasis by activating monocytes as well as endothelial cells. We find that neutrophil ectosomes have no proinflammatory activity on human macrophages as assessed by the release of interleukin 8 (IL-8) and tumor necrosis factor alpha (TNFalpha). On the contrary, ectosomes increase the release of transforming growth factor beta1 (TGFbeta1), suggesting that ectosomes down-modulate cellular activation in macrophages. Polymorphonuclear neutrophil (PMN) ectosomes are able to block inflammatory response of macrophages to zymosan and lipopolysaccharide (LPS). We show that an early-phase TGFbeta1 secretion and the exposure of phosphatidylserine on the surface of ectosomes independently contribute to this effect. Ectosome-cell contact was sufficient for their immunomodulatory function as shown by blocking phagocytosis with cytochalasin D. Thus, neutrophils release potent anti-inflammatory effectors, in the form of ectosomes, at the earliest stage of inflammation, already providing a drive to its resolution.