A versatile CRISPR-Cas13d platform for multiplexed transcriptomic regulation and metabolic engineering in primary human T cells.

CRISPR technologies have begun to revolutionize T cell therapies; however, conventional CRISPR-Cas9 genome-editing tools are limited in their safety, efficacy, and scope. To address these challenges, we developed multiplexed effector guide arrays (MEGA), a platform for programmable and scalable regulation of the T cell transcriptome using the RNA-guided, RNA-targeting activity of CRISPR-Cas13d. MEGA enables quantitative, reversible, and massively multiplexed gene knockdown in primary human T cells without targeting or cutting genomic DNA. Applying MEGA to a model of CAR T cell exhaustion, we robustly suppressed inhibitory receptor upregulation and uncovered paired regulators of T cell function through combinatorial CRISPR screening. We additionally implemented druggable regulation of MEGA to control CAR activation in a receptor-independent manner. Lastly, MEGA enabled multiplexed disruption of immunoregulatory metabolic pathways to enhance CAR T cell fitness and anti-tumor activity in vitro and in vivo. MEGA offers a versatile synthetic toolkit for applications in cancer immunotherapy and beyond.

Immunization of Vγ2Vδ2 T cells programs sustained effector memory responses that control tuberculosis in nonhuman primates.

Tuberculosis (TB) remains a leading killer among infectious diseases, and a better TB vaccine is urgently needed. The critical components and mechanisms of vaccine-induced protection against Mycobacterium tuberculosis (Mtb) remain incompletely defined. Our previous studies demonstrate that Vγ2Vδ2 T cells specific for (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) phosphoantigen are unique in primates as multifunctional effectors of immune protection against TB infection. Here, we selectively immunized Vγ2Vδ2 T cells and assessed the effect on infection in a rhesus TB model. A single respiratory vaccination of macaques with an HMBPP-producing attenuated Listeria monocytogenes (Lm ΔactA prfA*) caused prolonged expansion of HMBPP-specific Vγ2Vδ2 T cells in circulating and pulmonary compartments. This did not occur in animals similarly immunized with an Lm ΔgcpE strain, which did not produce HMBPP. Lm ΔactA prfA* vaccination elicited increases in Th1-like Vγ2Vδ2 T cells in the airway, and induced containment of TB infection after pulmonary challenge. The selective immunization of Vγ2Vδ2 T cells reduced lung pathology and mycobacterial dissemination to extrapulmonary organs. Vaccine effects coincided with the fast-acting memory-like response of Th1-like Vγ2Vδ2 T cells and tissue-resident Vγ2Vδ2 effector T cells that produced both IFN-γ and perforin and inhibited intracellular Mtb growth. Furthermore, selective immunization of Vγ2Vδ2 T cells enabled CD4+ and CD8+ T cells to mount earlier pulmonary Th1 responses to TB challenge. Our findings show that selective immunization of Vγ2Vδ2 T cells can elicit fast-acting and durable memory-like responses that amplify responses of other T cell subsets, and provide an approach to creating more effective TB vaccines.

Adoptive Transfer of Phosphoantigen-Specific γδ T Cell Subset Attenuates Mycobacterium tuberculosis Infection in Nonhuman Primates.

The dominant Vγ2Vδ2 T cell subset recognizes phosphoantigen and exists only in humans and nonhuman primates. Despite the discovery of γδ T cells >30 y ago, a proof-of-concept study has not been done to prove the principle that the Vγ2Vδ2 T cell subset is protective against Mycobacterium tuberculosis and other infections. In this study, we used an adoptive cell-transfer strategy to define the protective role of Vγ2Vδ2 T cells in a primate tuberculosis (TB) model. Vγ2Vδ2 T cells for adoptive transfer displayed central/effector memory and mounted effector functions, including the production of anti-M. tuberculosis cytokines and inhibition of intracellular mycobacteria. They also expressed CXCR3/CCR5/LFA-1 trafficking/tissue-resident phenotypes and consistently trafficked to the airway, where they remained detectable from 6 h through 7 d after adoptive transfer. Interestingly, the test group of macaques receiving transfer of Vγ2Vδ2 T cells at weeks 1 and 3 after high-dose (500 CFU) M. tuberculosis infection exhibited significantly lower levels of M. tuberculosis infection burdens in lung lobes and extrapulmonary organs than did the control groups receiving PBLs or saline. Consistently, adoptive transfer of Vγ2Vδ2 T cells attenuated TB pathology and contained lesions primarily in the infection site of the right caudal lung lobe, with no or reduced TB dissemination to other lobes, spleen, or liver/kidney; in contrast, the controls showed widespread TB dissemination. The proof-of-concept finding supports the view that the dominant Vγ2Vδ2 T cell subset may be included in the rational design of a TB vaccine or host-directed therapy.

Pathogenic Events in a Nonhuman Primate Model of Oral Poliovirus Infection Leading to Paralytic Poliomyelitis.

Despite a great deal of prior research, the early pathogenic events in natural oral poliovirus infection remain poorly defined. To establish a model for study, we infected 39 macaques by feeding them single high doses of the virulent Mahoney strain of wild type 1 poliovirus. Doses ranging from 107 to 109 50% tissue culture infective doses (TCID50) consistently infected all the animals, and many monkeys receiving 108 or 109 TCID50 developed paralysis. There was no apparent difference in the susceptibilities of the three macaque species (rhesus, cynomolgus, and bonnet) used. Virus excretion in stool and nasopharynges was consistently observed, with occasional viremia, and virus was isolated from tonsils, gut mucosa, and draining lymph nodes. Viral replication proteins were detected in both epithelial and lymphoid cell populations expressing CD155 in the tonsil and intestine, as well as in spinal cord neurons. Necrosis was observed in these three cell types, and viral replication in the tonsil/gut was associated with histopathologic destruction and inflammation. The sustained response of neutralizing antibody correlated temporally with resolution of viremia and termination of virus shedding in oropharynges and feces. For the first time, this model demonstrates that early in the infectious process, poliovirus replication occurs in both epithelial cells (explaining virus shedding in the gastrointestinal tract) and lymphoid/monocytic cells in tonsils and Peyer's patches (explaining viremia), extending previous studies of poliovirus pathogenesis in humans. Because the model recapitulates human poliovirus infection and poliomyelitis, it can be used to study polio pathogenesis and to assess the efficacy of candidate antiviral drugs and new vaccines.IMPORTANCE Early pathogenic events of poliovirus infection remain largely undefined, and there is a lack of animal models mimicking natural oral human infection leading to paralytic poliomyelitis. All 39 macaques fed with single high doses ranging from 107 to 109 TCID50 Mahoney type 1 virus were infected, and many of the monkeys developed paralysis. Virus excretion in stool and nasopharynges was consistently observed, with occasional viremia; tonsil, mesentery lymph nodes, and intestinal mucosa served as major target sites of viral replication. For the first time, this model demonstrates that early in the infectious process, poliovirus replication occurs in both epithelial cells (explaining virus shedding in the gastrointestinal tract) and lymphoid/monocytic cells in tonsils and Peyer's patches (explaining viremia), thereby supplementing historical reconstructions of poliovirus pathogenesis. Because the model recapitulates human poliovirus infection and poliomyelitis, it can be used to study polio pathogenesis, candidate antiviral drugs, and the efficacy of new vaccines.

Long noncoding RNA derived from CD244 signaling epigenetically controls CD8+ T-cell immune responses in tuberculosis infection.

Molecular mechanisms for T-cell immune responses modulated by T cell-inhibitory molecules during tuberculosis (TB) infection remain unclear. Here, we show that active human TB infection up-regulates CD244 and CD244 signaling-associated molecules in CD8(+) T cells and that blockade of CD244 signaling enhances production of IFN-γ and TNF-α. CD244 expression/signaling in TB correlates with high levels of a long noncoding RNA (lncRNA)-BC050410 [named as lncRNA-AS-GSTT1(1-72) or lncRNA-CD244] in the CD244(+)CD8(+) T-cell subpopulation. CD244 signaling drives lncRNA-CD244 expression via sustaining a permissive chromatin state in the lncRNA-CD244 locus. By recruiting polycomb protein enhancer of zeste homolog 2 (EZH2) to infg/tnfa promoters, lncRNA-CD244 mediates H3K27 trimethylation at infg/tnfa loci toward repressive chromatin states and inhibits IFN-γ/TNF-α expression in CD8(+) T cells. Such inhibition can be reversed by knock down of lncRNA-CD244. Interestingly, adoptive transfer of lncRNA-CD244-depressed CD8(+) T cells to Mycobacterium tuberculosis (MTB)-infected mice reduced MTB infection and TB pathology compared with lncRNA-CD244-expressed controls. Thus, this work uncovers previously unidentified mechanisms in which T cell-inhibitory signaling and lncRNAs regulate T-cell responses and host defense against TB infection.

Selective Destruction of Interleukin 23-Induced Expansion of a Major Antigen-Specific γδ T-Cell Subset in Patients With Tuberculosis.

A loss of antigen-specific T-cell responses due to defective cytokine signaling during infections has not been reported. We hypothesize that tuberculosis can destroy signaling effects of selective cytokine(s) and induce exhaustion of antigen-specific T cells. To test this hypothesis, mechanistic studies were performed to examine whether and how tuberculosis blocked interleukin 23 (IL-23) and interleukin 2 (IL-2) signaling effects on a major human γδ T-cell subpopulation, phosphoantigen HMBPP-specific Vγ2Vδ2 T cells. IL-23 and IL-2 significantly expanded HMBPP-stimulated Vγ2Vδ2 T cells from subjects with latent tuberculosis infection, and IL-2 synergized the effect of IL-23. IL-23-induced expansion of Vγ2Vδ2 T cells involved STAT3. Surprisingly, patients with tuberculosis exhibited a selective destruction of IL-23-induced expansion of these cells. The tuberculosis-driven destruction of IL-23 signaling coincided with decreases of expression and phosphorylation of STAT3. Interestingly, impairing of STAT3 was linked to marked increases in the microRNAs (miRNAs) hsa-miR-337-3p and hsa-miR-125b-5p in Vγ2Vδ2 T cells from patients with tuberculosis. Downregulation of hsa-miR-337-3p and hsa-miR-125b-5p by miRNA sponges improved IL-23-mediated expansion of Vγ2Vδ2 T cells and restored the ability of these cells to produce anti-tuberculosis cytokines. These results support our hypothesis that tuberculosis can selectively impair a cytokine effect while sparing another and can induce exhaustion of T cells in response to the respective cytokine.

Gram-positive pneumonia augments non-small cell lung cancer metastasis via host toll-like receptor 2 activation.

Surgical resection of early stage nonsmall cell lung cancer (NSCLC) is necessary for cure. However, rates of postoperative bacterial pneumonias remain high and may confer an increased risk for metastasis. Toll-like receptors (TLRs) mediate the inflammatory cascade by recognizing microbial products at the surface of numerous cell types in the lung; however, little is known about how host TLRs influence NSCLC metastasis. TLR2 recognizes gram-positive bacterial cell wall components activating innate immunity. We demonstrate that lower respiratory tract infection with Streptococcus pneumonia augments the formation of murine H59 NSCLC liver metastases in C57BL/6 mice through host TLR2 activation. Infected mice demonstrate increased H59 and human A549 NSCLC adhesion to hepatic sinusoids in vivo compared with noninfected controls, a response that is significantly diminished in TLR2 knock-out mice. Intra-tracheal injection of purified TLR2 ligand lipoteichoic acid into mice similarly augments in vivo adhesion of H59 cells to hepatic sinusoids. Additionally, H59 and A549 NSCLC cells incubated with bronchoepithelial conditioned media show increased cell adhesion to extracellular matrix components in vitro and hepatic sinusoids in vivo in a manner that is dependent on bronchoepithelial TLR2 activation and interleukin-6 secretion. TLR2 is therefore a potential therapeutic target for gram-positive pneumonia-driven NSCLC metastasis.

Comparison of pregabalin with pramipexole for restless legs syndrome.

BACKGROUND: Dopaminergic medications relieve symptoms of the restless legs syndrome (RLS) but have the potential to cause iatrogenic worsening (augmentation) of RLS with long-term treatment. Pregabalin may be an effective alternative. METHODS: In this 52-week, randomized, double-blind trial, we assessed efficacy and augmentation in patients with RLS who were treated with pregabalin as compared with placebo and pramipexole. Patients were randomly assigned to receive 52 weeks of treatment with pregabalin at a dose of 300 mg per day or pramipexole at a dose of 0.25 mg or 0.5 mg per day or 12 weeks of placebo followed by 40 weeks of randomly assigned active treatment. The primary analyses involved a comparison of pregabalin and placebo over a period of 12 weeks with use of the International RLS (IRLS) Study Group Rating Scale (on which the score ranges from 0 to 40, with a higher score indicating more severe symptoms), the Clinical Global Impression of Improvement scale (which was used to assess the proportion of patients with symptoms that were "very much improved" or "much improved"), and a comparison of rates of augmentation with pregabalin and pramipexole over a period of 40 or 52 weeks of treatment. RESULTS: A total of 719 participants received daily treatment, 182 with 300 mg of pregabalin, 178 with 0.25 mg of pramipexole, 180 with 0.5 mg of pramipexole, and 179 with placebo. Over a period of 12 weeks, the improvement (reduction) in mean scores on the IRLS scale was greater, by 4.5 points, among participants receiving pregabalin than among those receiving placebo (P<0.001), and the proportion of patients with symptoms that were very much improved or much improved was also greater with pregabalin than with placebo (71.4% vs. 46.8%, P<0.001). The rate of augmentation over a period of 40 or 52 weeks was significantly lower with pregabalin than with pramipexole at a dose of 0.5 mg (2.1% vs. 7.7%, P=0.001) but not at a dose of 0.25 mg (2.1% vs. 5.3%, P=0.08). There were six cases of suicidal ideation in the group receiving pregabalin, three in the group receiving 0.25 mg of pramipexole, and two in the group receiving 0.5 mg of pramipexole. CONCLUSIONS: Pregabalin provided significantly improved treatment outcomes as compared with placebo, and augmentation rates were significantly lower with pregabalin than with 0.5 mg of pramipexole. (Funded by Pfizer; ClinicalTrials.gov number, NCT00806026.).

Increasing resident utilization and recognition of the critical view of safety during laparoscopic cholecystectomy: a pilot study from an academic medical center.

BACKGROUND: Laparoscopic cholecystectomy (LC) is a commonly performed surgical procedure; however, it is associated with an increased rate of bile duct injury (BDI) when compared to the open approach. The critical view of safety (CVS) provides a secure method of ductal identification to help avoid BDI. CVS is not universally utilized by practicing surgeons and/or taught to surgical residents. We aimed to pilot a safe cholecystectomy curriculum to demonstrate that educational interventions could improve resident adherence to and recognition of the CVS during LC. METHODS: Forty-three general surgery residents at Thomas Jefferson University Hospital were prospectively studied. Fifty-one consecutive LC cases were recorded during the pre-intervention period, while the residents were blinded to the outcome measured (CVS score). As an intervention, a comprehensive lecture on safe cholecystectomy was given to all residents. Fifty consecutive LC cases were recorded post-intervention, while the residents were empowered to "time-out" and document the CVS with a doublet photograph. Two independent surgeons scored the videos and photographs using a 6-point scale. Residents were surveyed pre- and post-intervention to determine objective knowledge and self-reported comfort using a 5-point Likert scale. RESULTS: In the 18-week study period, 101 consecutive LCs were adequately captured and included (51 pre-intervention, 50 post-intervention). Patient demographics and clinical data were similar. The mean CVS score improved from 2.3 to 4.3 (p < 0.001). The number of videos with CVS score >4 increased from 15.7 to 52 % (p < 0.001). There was strong inter-observer agreement between reviewers. The pre- and post-intervention questionnaire response rates were 90.7 and 83.7 %, respectively. A greater number of residents correctly identified all criteria of the CVS post-intervention (41-93 %, p < 0.001) and offered appropriate bailout techniques (77-94 %, p < 0.001). Residents strongly agreed that the CVS education should be included in general surgery residency curriculum (mean Likert score = 4.71, SD = 0.54). Residents also agreed that they are more comfortable with their LC skills after the intervention (4.27, σ = 0.83). CONCLUSION: The combination of focused education along with intraoperative time-out significantly improved CVS scores and knowledge during LC in our institution.

Salmon Bias and Preterm Birth Among Western Immigrants in China.

Introduction Immigrants from Western industrialized countries are rarely found in immigrant studies. Our primary objective was to calculate the rate of cesarean delivery, 5-min Apgar score <7, and preterm birth among Chinese and Western women. Our secondary objective was to examine whether there are significant differences in terms of risk factors between Western immigrants who chose to deliver in their country of citizenship compared to those who chose to deliver in China. Methods Single-center retrospective cohort study in Shanghai, China. Multivariate logistic regression models used delivery outcome, and place of delivery (China vs. country of citizenship) as outcome variables. Results Preterm birth occurred at a rate of 3.82% among Chinese citizens, 4.12% among Chinese-born Western citizens, and 6.54% among non-Chinese-born Western citizens. After adjustment, preterm birth <37 weeks was more frequent among non-Chinese-born Western citizens compared with Chinese citizens, with an odds ratio of 1.82 (Confidence Interval 1.20-2.78), p = 0.005. Variables statistically associated with giving birth in China were maternal age ≥35 years and being Chinese-born Western, as well as the absence of medical or obstetrical conditions. Discussion Western immigrants have overall good obstetrical outcomes in China, and this could be partly explained by selective immigration, but also by the Salmon bias, as women with risk factors tend to return to their country of citizenship for the delivery. However, the preterm birth rate was higher among Western women than in their Chinese counterparts, and further research is needed.

Vaginal birth after caesarean delivery in Chinese women and Western immigrants in Shanghai.

Recent studies show a steep rise in caesarean sections in China. Most couples are now eligible to apply for a second child. This retrospective cohort study compares the prevalence of trial of labour and vaginal birth after caesarean section among Chinese and foreign women in Shanghai. In total, 135 of 368 women underwent trial of labour (36.68%), and of those, 77 (57.04%) had a vaginal birth. After inclusion in a multivariate model, factors associated with trial of labour were maternal age <35 years with an adjusted odds ratio of 2.58 (1.49-4.46), absence of a history of ≥3 abortions (2.22 (1.08-4.57)), and European citizenship (1.94 (1.05-3.59)). The prevalence of trial of labour and vaginal birth seems to mirror rates found in countries of origin, but despite a high rate of caesarean section, Chinese women had a higher rate of vaginal birth after caesarean section than North American and Australian women, in particular. Impact statement What is already known on this subject: Caesarean section (CS) rates are rising worldwide. Repeat CS contributes largely to these rates, although vaginal birth after CS (VBAC) rates varies widely between countries. What the results of this study add: North American and Australian women who deliver in Shanghai have low rates of attempted trial of labour after CS (TOLAC) and VBAC, with European women having the highest rate of TOLAC, followed by Chinese women. Implications for clinical practice and/or further research: These findings might reflect different levels of acceptance in line with respective national trends. Studies evaluating the influence of cultural norms on birth preferences after CS are needed. Further research is also needed to assess the overall acceptance of TOLAC in the context of the softening of the one-child policy in China.

Th17-related cytokines contribute to recall-like expansion/effector function of HMBPP-specific Vγ2Vδ2 T cells after Mycobacterium tuberculosis infection or vaccination.

Whether cytokines can influence the adaptive immune response by antigen-specific γδ T cells during infections or vaccinations remains unknown. We previously demonstrated that, during BCG/Mycobacterium tuberculosis (Mtb) infections, Th17-related cytokines markedly upregulated when phosphoantigen-specific Vγ2Vδ2 T cells expanded. In this study, we examined the involvement of Th17-related cytokines in the recall-like responses of Vγ2Vδ2 T cells following Mtb infection or vaccination against TB. Treatment with IL-17A/IL-17F or IL-22 expanded phosphoantigen 4-hydroxy-3-methyl-but-enyl pyrophosphate (HMBPP)-stimulated Vγ2Vδ2 T cells from BCG-vaccinated macaques but not from naïve animals, and IL-23 induced greater expansion than the other Th17-related cytokines. Consistently, Mtb infection of macaques also enhanced the ability of IL-17/IL-22 or IL-23 to expand HMBPP-stimulated Vγ2Vδ2 T cells. When evaluating IL-23 signaling as a prototype, we found that HMBPP/IL-23-expanded Vγ2Vδ2 T cells from macaques infected with Mtb or vaccinated with BCG or Listeria ΔactA prfA*-ESAT6/Ag85B produced IL-17, IL-22, IL-2, and IFN-γ. Interestingly, HMBPP/IL-23-induced production of IFN-γ in turn facilitated IL-23-induced expansion of HMBPP-activated Vγ2Vδ2 T cells. Furthermore, HMBPP/IL-23-induced proliferation of Vγ2Vδ2 T cells appeared to require APC contact and involve the conventional and novel protein kinase C signaling pathways. These findings suggest that Th17-related cytokines can contribute to recall-like expansion and effector function of Ag-specific γδ T cells after infection or vaccination.

CD4+ T cells contain early extrapulmonary tuberculosis (TB) dissemination and rapid TB progression and sustain multieffector functions of CD8+ T and CD3- lymphocytes: mechanisms of CD4+ T cell immunity.

The possibility that CD4(+) T cells can act as "innate-like" cells to contain very early Mycobacterium tuberculosis dissemination and function as master helpers to sustain multiple effector functions of CD8(+) T cells and CD3(-) lymphocytes during development of adaptive immunity against primary tuberculosis (TB) has not been demonstrated. We showed that pulmonary M. tuberculosis infection of CD4-depleted macaques surprisingly led to very early extrapulmonary M. tuberculosis dissemination, whereas CD4 deficiency clearly resulted in rapid TB progression. CD4 depletion during M. tuberculosis infection revealed the ability of CD8(+) T cells to compensate and rapidly differentiate to Th17-like/Th1-like and cytotoxic-like effectors, but these effector functions were subsequently unsustainable due to CD4 deficiency. Whereas CD3(-) non-T lymphocytes in the presence of CD4(+) T cells developed predominant Th22-like and NK-like (perforin production) responses to M. tuberculosis infection, CD4 depletion abrogated these Th22-/NK-like effector functions and favored IL-17 production by CD3(-) lymphocytes. CD4-depleted macaques exhibited no or few pulmonary T effector cells constitutively producing IFN-γ, TNF-α, IL-17, IL-22, and perforin at the endpoint of more severe TB, but they presented pulmonary IL-4(+) T effectors. TB granulomas in CD4-depleted macaques contained fewer IL-22(+) and perforin(+) cells despite the presence of IL-17(+) and IL-4(+) cells. These results implicate a previously unknown innate-like ability of CD4(+) T cells to contain extrapulmonary M. tuberculosis dissemination at very early stage. Data also suggest that CD4(+) T cells are required to sustain multiple effector functions of CD8(+) T cells and CD3(-) lymphocytes and to prevent rapid TB progression during M. tuberculosis infection of nonhuman primates.

Gram negative bacteria increase non-small cell lung cancer metastasis via Toll-like receptor 4 activation and mitogen-activated protein kinase phosphorylation.

Surgery is required for the curative treatment of lung cancer but is associated with high rates of postoperative pneumonias predominantly caused by gram negative bacteria. Recent evidence suggests that these severe infectious complications may decrease long term survival after hospital discharge via cancer recurrence, but the mechanism is unclear. Lung cancer cells have recently been demonstrated to express Toll-like receptors (TLR) that mediate pathogen recognition. We hypothesized that incubation of non-small cell lung cancer (NSCLC) cells with heat-inactivated Escherichia coli can augment cancer cell adhesion, migration and metastasis via TLR4 signaling. Incubation of murine and human NSCLC cells with E. coli increased in vitro cell adhesion to collagen I, collagen IV and fibronectin, and enhanced in vitro migration. Using hepatic intravital microscopy, we demonstrated that NSCLC cells have increased in vivo adhesion to hepatic sinusoids after coincubation with gram negative bacteria. These enhanced cell adhesion and migration phenotypes following incubation with E. coli were attenuated at three levels: inhibition of TLR4 (Eritoran), p38 MAPK (BIRB0796) and ERK1/2 phosphorylation (PD184352). Incubation of murine NSCLC cells in vitro with E. coli prior to intrasplenic injection significantly augmented formation of in vivo hepatic metastases 2 weeks later. This increase was abrogated by NSCLC TLR4 blockade using Eritoran. TLR4 represents a potential therapeutic target to help prevent severe postoperative infection driven cancer metastasis.

Phosphoantigen/IL2 expansion and differentiation of Vγ2Vδ2 T cells increase resistance to tuberculosis in nonhuman primates.

Dominant Vγ2Vδ2 T-cell subset exist only in primates, and recognize phosphoantigen from selected pathogens including M. tuberculosis(Mtb). In vivo function of Vγ2Vδ2 T cells in tuberculosis remains unknown. We conducted mechanistic studies to determine whether earlier expansion/differentiation of Vγ2Vδ2 T cells during Mtb infection could increase immune resistance to tuberculosis in macaques. Phosphoantigen/IL-2 administration specifically induced major expansion and pulmonary trafficking/accumulation of phosphoantigen-specific Vγ2Vδ2 T cells, significantly reduced Mtb burdens and attenuated tuberculosis lesions in lung tissues compared to saline/BSA or IL-2 controls. Expanded Vγ2Vδ2 T cells differentiated into multifunctional effector subpopulations capable of producing anti-TB cytokines IFNγ, perforin and granulysin, and co-producing perforin/granulysin in lung tissue. Mechanistically, perforin/granulysin-producing Vγ2Vδ2 T cells limited intracellular Mtb growth, and macaque granulysin had Mtb-bactericidal effect, and inhibited intracellular Mtb in presence of perforin. Furthermore, phosphoantigen/IL2-expanded Vγ2Vδ2 T effector cells produced IL-12, and their expansion/differentiation led to enhanced pulmonary responses of peptide-specific CD4+/CD8+ Th1-like cells. These results provide first in vivo evidence implicating that early expansion/differentiation of Vγ2Vδ2 T effector cells during Mtb infection increases resistance to tuberculosis. Thus, data support a rationale for conducting further studies of the γδ T-cell-targeted treatment of established TB, which might ultimately help explore single or adjunctive phosphoantigen expansion of Vγ2Vδ2 T-cell subset as intervention of MDR-tuberculosis or HIV-related tuberculosis.

Pregabalin does not affect sperm production in healthy volunteers: a randomized, double-blind, placebo-controlled, noninferiority study.

OBJECTIVE: The primary objective of this study was to compare the effects of pregabalin and placebo on sperm concentration in healthy male subjects. Changes in follicle-stimulating hormone (FSH), testosterone, sperm motility, semen volume, and sperm morphology were also examined. METHODS: This was a phase 4, multicenter, double-blind, randomized, placebo-controlled, noninferiority study. A 12-week treatment period (placebo or 600 mg/day of pregabalin) was followed by a 1-week taper period and a 13-week washout period. The primary outcome measure was the percentage of subjects with a ≥ 50% reduction from baseline in sperm concentration at End of Study. RESULTS: One hundred and nine subjects received placebo and 111 subjects received pregabalin. The difference between placebo and pregabalin with respect to the percentage of subjects with a ≥ 50% reduction from baseline in sperm concentration at End of Study was 6% (95% CI: -2.29 to 14.3%). Noninferiority of pregabalin compared to placebo was declared as the upper bound of the 95% CI was less than the prespecified noninferiority margin of 20%. There were no significant differences between placebo and pregabalin groups with respect to their effects on FSH, testosterone, or sperm motility. Changes in semen volume and sperm morphology were numerically similar in both treatment groups. Adverse events were consistent with the known safety profile of pregabalin. Treatment with 600 mg/day pregabalin for 12 weeks does not adversely affect spermatogenesis or serum levels of FSH and testosterone in healthy males.

Tim-3-expressing CD4+ and CD8+ T cells in human tuberculosis (TB) exhibit polarized effector memory phenotypes and stronger anti-TB effector functions.

T-cell immune responses modulated by T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) during Mycobacterium tuberculosis (Mtb) infection in humans remain poorly understood. Here, we found that active TB patients exhibited increases in numbers of Tim-3-expressing CD4(+) and CD8(+) T cells, which preferentially displayed polarized effector memory phenotypes. Consistent with effector phenotypes, Tim-3(+)CD4(+) and Tim-3(+)CD8(+) T-cell subsets showed greater effector functions for producing Th1/Th22 cytokines and CTL effector molecules than Tim-3(-) counterparts, and Tim-3-expressing T cells more apparently limited intracellular Mtb replication in macrophages. The increased effector functions for Tim-3-expressing T cells consisted with cellular activation signaling as Tim-3(+)CD4(+) and Tim-3(+)CD8(+) T-cell subsets expressed much higher levels of phosphorylated signaling molecules p38, stat3, stat5, and Erk1/2 than Tim-3- controls. Mechanistic experiments showed that siRNA silencing of Tim-3 or soluble Tim-3 treatment interfering with membrane Tim-3-ligand interaction reduced de novo production of IFN-γ and TNF-α by Tim-3-expressing T cells. Furthermore, stimulation of Tim-3 signaling pathways by antibody cross-linking of membrane Tim-3 augmented effector function of IFN-γ production by CD4(+) and CD8(+) T cells, suggesting that Tim-3 signaling helped to drive stronger effector functions in active TB patients. This study therefore uncovered a previously unknown mechanism for T-cell immune responses regulated by Tim-3, and findings may have implications for potential immune intervention in TB.

Multieffector-functional immune responses of HMBPP-specific Vγ2Vδ2 T cells in nonhuman primates inoculated with Listeria monocytogenes ΔactA prfA*.

Although Listeria monocytogenes can induce systemic infection causing spontaneous abortion, septicemia, and meningitis, studies have not been performed to investigate human anti-L. monocytogenes immune responses, including those of Ag-specific Vγ2Vδ2 T cells, a dominant human γδ T cell subset. L. monocytogenes is the only pathogen known to possess both the mevalonate and non-mevalonate isoprenoid biosynthesis pathways that produce metabolic phosphates or phosphoantigens activating human Vγ2Vδ2 T cells, making it interesting to explore in vivo anti-L. monocytogenes immune responses of Vγ2Vδ2 T cells. In this study, we demonstrated that subclinical systemic L. monocytogenes infection of rhesus macaques via parenteral inoculation or vaccination with an attenuated Listeria strain induced multieffector-functional immune responses of phosphoantigen-specific Vγ2Vδ2 T cells. Subclinical systemic infection and reinfection with attenuated L. monocytogenes uncovered the ability of Vγ2Vδ2 T cells to mount expansion and adaptive or recall-like expansion. Expanded Vγ2Vδ2 T cells could traffic to and accumulate in the pulmonary compartment and intestinal mucosa. Expanded Vγ2Vδ2 T cells could evolve into effector cells producing IFN-γ, TNF-α, IL-4, IL-17, or perforin after L. monocytogenes infection, and some effector Vγ2Vδ2 T cells could coproduce IL-17 and IFN-γ, IL-4 and IFN-γ, or TNF-α and perforin. Surprisingly, in vivo-expanded Vγ2Vδ2 T effector cells in subclinical L. monocytogenes infection could directly lyse L. monocytogenes-infected target cells and inhibit intracellular L. monocytogenes bacteria. Thus, we present the first demonstration, to our knowledge, of multieffector-functional Vγ2Vδ2 T cell responses against L. monocytogenes.