Helper T cell immunity in humans with inherited CD4 deficiency.

CD4+ T cells are vital for host defense and immune regulation. However, the fundamental role of CD4 itself remains enigmatic. We report seven patients aged 5-61 years from five families of four ancestries with autosomal recessive CD4 deficiency and a range of infections, including recalcitrant warts and Whipple's disease. All patients are homozygous for rare deleterious CD4 variants impacting expression of the canonical CD4 isoform. A shorter expressed isoform that interacts with LCK, but not HLA class II, is affected by only one variant. All patients lack CD4+ T cells and have increased numbers of TCRαß+CD4-CD8- T cells, which phenotypically and transcriptionally resemble conventional Th cells. Finally, patient CD4-CD8- αß T cells exhibit intact responses to HLA class II-restricted antigens and promote B cell differentiation in vitro. Thus, compensatory development of Th cells enables patients with inherited CD4 deficiency to acquire effective cellular and humoral immunity against an unexpectedly large range of pathogens. Nevertheless, CD4 is indispensable for protective immunity against at least human papillomaviruses and Trophyrema whipplei.

Memory-like Differentiation, Tumor-Targeting mAbs, and Chimeric Antigen Receptors Enhance Natural Killer Cell Responses to Head and Neck Cancer.

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) is an aggressive tumor with low response rates to frontline PD-1 blockade. Natural killer (NK) cells are a promising cellular therapy for T cell therapy-refractory cancers, but are frequently dysfunctional in patients with HNSCC. Strategies are needed to enhance NK cell responses against HNSCC. We hypothesized that memory-like (ML) NK cell differentiation, tumor targeting with cetuximab, and engineering with an anti-EphA2 (Erythropoietin-producing hepatocellular receptor A2) chimeric antigen receptor (CAR) enhance NK cell responses against HNSCC. EXPERIMENTAL DESIGN: We generated ML NK and conventional (c)NK cells from healthy donors, then evaluated their ability to produce IFNγ, TNF, degranulate, and kill HNSCC cell lines and primary HNSCC cells, alone or in combination with cetuximab, in vitro and in vivo using xenograft models. ML and cNK cells were engineered to express anti-EphA2 CAR-CD8A-41BB-CD3z, and functional responses were assessed in vitro against HNSCC cell lines and primary HNSCC tumor cells. RESULTS: Human ML NK cells displayed enhanced IFNγ and TNF production and both short- and long-term killing of HNSCC cell lines and primary targets, compared with cNK cells. These enhanced responses were further improved by cetuximab. Compared with controls, ML NK cells expressing anti-EphA2 CAR had increased IFNγ and cytotoxicity in response to EphA2+ cell lines and primary HNSCC targets. CONCLUSIONS: These preclinical findings demonstrate that ML differentiation alone or coupled with either cetuximab-directed targeting or EphA2 CAR engineering were effective against HNSCCs and provide the rationale for investigating these combination approaches in early phase clinical trials for patients with HNSCC.

T-BET and EOMES sustain mature human NK cell identity and antitumor function.

Since the T-box transcription factors (TFs) T-BET and EOMES are necessary for initiation of NK cell development, their ongoing requirement for mature NK cell homeostasis, function, and molecular programming remains unclear. To address this, T-BET and EOMES were deleted in unexpanded primary human NK cells using CRISPR/Cas9. Deleting these TFs compromised in vivo antitumor response of human NK cells. Mechanistically, T-BET and EOMES were required for normal NK cell proliferation and persistence in vivo. NK cells lacking T-BET and EOMES also exhibited defective responses to cytokine stimulation. Single-cell RNA-Seq revealed a specific T-box transcriptional program in human NK cells, which was rapidly lost following T-BET and EOMES deletion. Further, T-BET- and EOMES-deleted CD56bright NK cells acquired an innate lymphoid cell precursor-like (ILCP-like) profile with increased expression of the ILC-3-associated TFs RORC and AHR, revealing a role for T-box TFs in maintaining mature NK cell phenotypes and an unexpected role of suppressing alternative ILC lineages. Our study reveals the critical importance of sustained EOMES and T-BET expression to orchestrate mature NK cell function and identity.

Killer-Cell Immunoglobulin-like Receptor Diversity in an Admixed South American Population.

Natural Killer (NK) cells are innate immune cells that mediate antiviral and antitumor responses. NK cell activation and induction of effector functions are tightly regulated by the integration of activating and inhibitory receptors such as killer immunoglobulin-like receptors (KIR). KIR genes are characterized by a high degree of diversity due to presence or absence, gene copy number and allelic polymorphism. The aim of this study was to establish the distribution of KIR genes and genotypes, to infer the most common haplotypes in an admixed Colombian population and to compare these KIR gene frequencies with some Central and South American populations and worldwide. A total of 161 individuals from Medellin, Colombia were included in the study. Genomic DNA was used for KIR and HLA genotyping. We analyzed only KIR gene-content (presence or absence) based on PCR-SSO. The KIR genotype, most common haplotypes and combinations of KIR and HLA ligands frequencies were estimated according to the presence or absence of KIR and HLA genes. Dendrograms, principal component (PC) analysis and Heatmap analysis based on genetic distance were constructed to compare KIR gene frequencies among Central and South American, worldwide and Amerindian populations. The 16 KIR genes analyzed were distributed in 37 different genotypes and the 7 most frequent KIR inferred haplotypes. Importantly, we found three new genotypes not previously reported in any other ethnic group. Our genetic distance, PC and Heatmap analysis revealed marked differences in the distribution of KIR gene frequencies in the Medellin population compared to worldwide populations. These differences occurred mainly in the activating KIR isoforms, which are more frequent in our population, particularly KIR3DS1. Finally, we observed unique structural patterns of genotypes, which evidences the potential diversity and variability of this gene family in our population, and the need for exhaustive genetic studies to expand our understanding of the KIR gene complex in Colombian populations.

A novel fusion protein scaffold 18/12/TxM activates the IL-12, IL-15, and IL-18 receptors to induce human memory-like natural killer cells.

Natural killer (NK) cells are cytotoxic innate lymphoid cells that are emerging as a cellular immunotherapy for various malignancies. NK cells are particularly dependent on interleukin (IL)-15 for their survival, proliferation, and cytotoxic function. NK cells differentiate into memory-like cells with enhanced effector function after a brief activation with IL-12, IL-15, and IL-18. N-803 is an IL-15 superagonist composed of an IL-15 mutant (IL-15N72D) bound to the sushi domain of IL-15Rα fused to the Fc region of IgG1, which results in physiological trans-presentation of IL-15. Here, we describe the creation of a novel triple-cytokine fusion molecule, 18/12/TxM, using the N-803 scaffold fused to IL-18 via the IL-15N72D domain and linked to a heteromeric single-chain IL-12 p70 by the sushi domain of the IL-15Rα. This molecule displays trispecific cytokine activity through its binding and signaling through the individual cytokine receptors. Compared with activation with the individual cytokines, 18/12/TxM induces similar short-term activation and memory-like differentiation of NK cells on both the transcriptional and protein level and identical in vitro and in vivo anti-tumor activity. Thus, N-803 can be modified as a functional scaffold for the creation of cytokine immunotherapies with multiple receptor specificities to activate NK cells for adoptive cellular therapy.

Hematopoietic cell transplantation donor-derived memory-like NK cells functionally persist after transfer into patients with leukemia.

Natural killer (NK) cells are innate lymphoid cells that eliminate cancer cells, produce cytokines, and are being investigated as a nascent cellular immunotherapy. Impaired NK cell function, expansion, and persistence remain key challenges for optimal clinical translation. One promising strategy to overcome these challenges is cytokine-induced memory-like (ML) differentiation, whereby NK cells acquire enhanced antitumor function after stimulation with interleukin-12 (IL-12), IL-15, and IL-18. Here, reduced-intensity conditioning (RIC) for HLA-haploidentical hematopoietic cell transplantation (HCT) was augmented with same-donor ML NK cells on day +7 and 3 weeks of N-803 (IL-15 superagonist) to treat patients with relapsed/refractory acute myeloid leukemia (AML) in a clinical trial (NCT02782546). In 15 patients, donor ML NK cells were well tolerated, and 87% of patients achieved a composite complete response at day +28, which corresponded with clearing high-risk mutations, including TP53 variants. NK cells were the major blood lymphocytes for 2 months after HCT with 1104-fold expansion (over 1 to 2 weeks). Phenotypic and transcriptional analyses identified donor ML NK cells as distinct from conventional NK cells and showed that ML NK cells persisted for over 2 months. ML NK cells expressed CD16, CD57, and high granzyme B and perforin, along with a unique transcription factor profile. ML NK cells differentiated in patients had enhanced ex vivo function compared to conventional NK cells from both patients and healthy donors. Overall, same-donor ML NK cell therapy with 3 weeks of N-803 support safely augmented RIC haplo-HCT for AML.

Lung Epithelial Signaling Mediates Early Vaccine-Induced CD4+ T Cell Activation and Mycobacterium tuberculosis Control.

Tuberculosis (TB) is one of the leading causes of death due to a single infectious agent. The development of a TB vaccine that induces durable and effective immunity to Mycobacterium tuberculosis (Mtb) infection is urgently needed. Early and superior Mtb control can be induced in M. bovis Bacillus Calmette-Guérin (BCG)-vaccinated hosts when the innate immune response is targeted to generate effective vaccine-induced immunity. In the present study, we show that innate activation of DCs is critical for mucosal localization of clonally activated vaccine-induced CD4+ T cells in the lung and superior early Mtb control. In addition, our study reveals that Th1/Th17 cytokine axis play an important role in superior vaccine-induced immunity. Our studies also show that activation of the nuclear factor kappa-light-chain enhancer of activated B cell (NF-κß) pathway in lung epithelial cells is critical for the mucosal localization of activated vaccine-induced CD4+ T cells for rapid Mtb control. Thus, our study provides novel insights into the immune mechanisms that can overcome TB vaccine bottlenecks and provide early rapid Mtb control. IMPORTANCE Tuberculosis is a leading cause of death due to single infectious agent accounting 1.4 million deaths each year. The only licensed vaccine, BCG, is not effective due to variable efficacy. In our study, we determined the early immune events necessary for achieving complete protection in a BCG-vaccinated host. Our study reveals that innate activation of DCs can mediate superior and early Mtb control in BCG-vaccinated mice through lung epithelial cell signaling and localization of clonal activated, Mtb antigen-specific, cytokine-producing CD4+ T cells within the lung parenchyma and airways. Thus, our study provides novel insights into the immune mechanisms that can overcome TB vaccine bottlenecks and provide early rapid Mtb control.

Memory-like Differentiation Enhances NK Cell Responses to Melanoma.

PURPOSE: Treatment of advanced melanoma is a clinical challenge. Natural killer (NK) cells are a promising cellular therapy for T cell-refractory cancers, but are frequently deficient or dysfunctional in patients with melanoma. Thus, new strategies are needed to enhance NK-cell antitumor responses. Cytokine-induced memory-like (ML) differentiation overcomes many barriers in the NK-cell therapeutics field, resulting in potent cytotoxicity and enhanced cytokine production against blood cancer targets. However, the preclinical activity of ML NK against solid tumors remains largely undefined. EXPERIMENTAL DESIGN: Phenotypic and functional alterations of blood and advanced melanoma infiltrating NK cells were evaluated using mass cytometry. ML NK cells from healthy donors (HD) and patients with advanced melanoma were evaluated for their ability to produce IFNγ and kill melanoma targets in vitro and in vivo using a xenograft model. RESULTS: NK cells in advanced melanoma exhibited a decreased cytotoxic potential compared with blood NK cells. ML NK cells differentiated from HD and patients with advanced melanoma displayed enhanced IFNγ production and cytotoxicity against melanoma targets. This included ML differentiation enhancing melanoma patients' NK-cell responses against autologous targets. The ML NK-cell response against melanoma was partially dependent on the NKG2D- and NKp46-activating receptors. Furthermore, in xenograft NSG mouse models, human ML NK cells demonstrated superior control of melanoma, compared with conventional NK cells. CONCLUSIONS: Blood NK cells from allogeneic HD or patients with advanced melanoma can be differentiated into ML NK cells for use as a novel immunotherapeutic treatment for advanced melanoma, which warrants testing in early-phase clinical trials.

Combining AFM13, a Bispecific CD30/CD16 Antibody, with Cytokine-Activated Blood and Cord Blood-Derived NK Cells Facilitates CAR-like Responses Against CD30+ Malignancies.

PURPOSE: Natural killer (NK)-cell recognition and function against NK-resistant cancers remain substantial barriers to the broad application of NK-cell immunotherapy. Potential solutions include bispecific engagers that target NK-cell activity via an NK-activating receptor when simultaneously targeting a tumor-specific antigen, as well as enhancing functionality using IL12/15/18 cytokine pre-activation. EXPERIMENTAL DESIGN: We assessed single-cell NK-cell responses stimulated by the tetravalent bispecific antibody AFM13 that binds CD30 on leukemia/lymphoma targets and CD16A on various types of NK cells using mass cytometry and cytotoxicity assays. The combination of AFM13 and IL12/15/18 pre-activation of blood and cord blood-derived NK cells was investigated in vitro and in vivo. RESULTS: We found heterogeneity within AFM13-directed conventional blood NK cell (cNK) responses, as well as consistent AFM13-directed polyfunctional activation of mature NK cells across donors. NK-cell source also impacted the AFM13 response, with cNK cells from healthy donors exhibiting superior responses to those from patients with Hodgkin lymphoma. IL12/15/18-induced memory-like NK cells from peripheral blood exhibited enhanced killing of CD30+ lymphoma targets directed by AFM13, compared with cNK cells. Cord-blood NK cells preactivated with IL12/15/18 and ex vivo expanded with K562-based feeders also exhibited enhanced killing with AFM13 stimulation via upregulation of signaling pathways related to NK-cell effector function. AFM13-NK complex cells exhibited enhanced responses to CD30+ lymphomas in vitro and in vivo. CONCLUSIONS: We identify AFM13 as a promising combination with cytokine-activated adult blood or cord-blood NK cells to treat CD30+ hematologic malignancies, warranting clinical trials with these novel combinations.

Phase I Trial of N-803, an IL15 Receptor Agonist, with Rituximab in Patients with Indolent Non-Hodgkin Lymphoma.

PURPOSE: N-803 is an IL15 receptor superagonist complex, designed to optimize in vivo persistence and trans-presentation, thereby activating and expanding natural killer (NK) cells and CD8+ T cells. Monoclonal antibodies (mAbs) direct Fc receptor-bearing immune cells, including NK cells, to recognize and eliminate cancer targets. The ability of IL15R agonists to enhance tumor-targeting mAbs in patients has not been reported previously. PATIENTS AND METHODS: Relapsed/refractory patients with indolent non-Hodgkin lymphoma were treated with rituximab and intravenous or subcutaneous N-803 on an open-label, dose-escalation phase I study using a 3+3 design (NCT02384954). Primary endpoint was maximum tolerated dose. Immune correlates were performed using multidimensional analysis via mass cytometry and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) which simultaneously measures protein and single-cell RNA expression. RESULTS: This immunotherapy combination was safe and well tolerated and resulted in durable clinical responses including in rituximab-refractory patients. Subcutaneous N-803 plus rituximab induced sustained proliferation, expansion, and activation of peripheral blood NK cells and CD8 T cells, with increased NK cell and T cells present 8 weeks following last N-803 treatment. CITE-seq revealed a therapy-altered NK cell molecular program, including enhancement of AP-1 transcription factor. Furthermore, the monocyte transcriptional program was remodeled with enhanced MHC expression and antigen-presentation genes. CONCLUSIONS: N-803 combines with mAbs to enhance tumor targeting in patients, and warrants further investigation in combination with immunotherapies.

CAR-modified memory-like NK cells exhibit potent responses to NK-resistant lymphomas.

Natural killer (NK) cells are a promising cellular immunotherapy for cancer. Cytokine-induced memory-like (ML) NK cells differentiate after activation with interleukin-12 (IL-12), IL-15, and IL-18, exhibit potent antitumor responses, and safely induce complete remissions in patients with leukemia. However, many cancers are not fully recognized via NK cell receptors. Chimeric antigen receptors (CARs) have been used to enhance tumor-specific recognition by effector lymphocytes. We hypothesized that ML differentiation and CAR engineering would result in complementary improvements in NK cell responses against NK-resistant cancers. To test this idea, peripheral blood ML NK cells were modified to express an anti-CD19 CAR (19-CAR-ML), which displayed significantly increased interferon γ production, degranulation, and specific killing against NK-resistant lymphoma lines and primary targets compared with nonspecific control CAR-ML NK cells or conventional CAR NK cells. The 19-CAR and ML responses were synergistic and CAR specific and required immunoreceptor tyrosine-based activation motif signaling. Furthermore, 19-CAR-ML NK cells generated from lymphoma patients exhibited improved responses against their autologous lymphomas. 19-CAR-ML NK cells controlled lymphoma burden in vivo and improved survival in human xenograft models. Thus, CAR engineering of ML NK cells enhanced responses against resistant cancers and warrants further investigation, with the potential to broaden ML NK cell recognition against a variety of NK cell-resistant tumors.

Stage-Specific Requirement for Eomes in Mature NK Cell Homeostasis and Cytotoxicity.

Natural killer (NK) cells are cytotoxic innate lymphoid cells (ILCs) that mediate antiviral and antitumor responses and require the transcriptional regulator Eomesodermin (Eomes) for early development. However, the role of Eomes and its molecular program in mature NK cell biology is unclear. To address this, we develop a tamoxifen-inducible, type-1-ILC-specific (Ncr1-targeted) cre mouse and combine this with Eomes-floxed mice. Eomes deletion after normal NK cell ontogeny results in a rapid loss of NK cells (but not ILC1s), with a particularly profound effect on penultimately mature stage III NK cells. Mechanisms responsible for stage III reduction include increased apoptosis and impaired maturation from stage II precursors. Induced Eomes deletion also decreases NK cell cytotoxicity and abrogates in vivo rejection of major histocompatibility complex (MHC)-class-I-deficient cells. However, other NK cell functional responses, and stage IV NK cells, are largely preserved. These data indicate that mature NK cells have distinct Eomes-dependent and -independent stages.

Friend or Foe: The Protective and Pathological Roles of Inducible Bronchus-Associated Lymphoid Tissue in Pulmonary Diseases.

Inducible bronchus-associated lymphoid tissue (iBALT) is a tertiary lymphoid structure that resembles secondary lymphoid organs. iBALT is induced in the lung in response to Ag exposure. In some cases, such as infection with Mycobacterium tuberculosis, the formation of iBALT structure is indicative of an effective protective immune response. However, with persistent exposure to Ags during chronic inflammation, allergy, or autoimmune diseases, iBALT may be associated with exacerbation of inflammation. iBALT is characterized by well-organized T and B areas enmeshed with conventional dendritic cells, follicular dendritic cells, and stromal cells, usually located surrounding airways or blood vessels. Several of the molecular signals and cellular contributors that mediate formation of iBALT structures have been recently identified. This review will outline the recent findings associated with the formation and maintenance of iBALT and their contributions toward a protective or pathogenic function in pulmonary disease outcome.

Publisher Correction: Mycobacterium tuberculosis carrying a rifampicin drug resistance mutation reprograms macrophage metabolism through cell wall lipid changes.

In the version of this Letter originally published, in Fig. 2d, in the third graph, the label for the y axis was incorrect as 'TNF-α (pg ml-1)'; it should have read 'IL-1ß (pg ml-1)'. This has now been corrected.

Mycobacterium tuberculosis carrying a rifampicin drug resistance mutation reprograms macrophage metabolism through cell wall lipid changes.

Tuberculosis is a significant global health threat, with one-third of the world's population infected with its causative agent Mycobacterium tuberculosis (Mtb). The emergence of multidrug-resistant (MDR) Mtb that is resistant to the frontline anti-tubercular drugs rifampicin and isoniazid forces treatment with toxic second-line drugs. Currently, ~4% of new and ~21% of previously treated tuberculosis cases are either rifampicin-drug-resistant or MDR Mtb infections1. The specific molecular host-pathogen interactions mediating the rapid worldwide spread of MDR Mtb strains remain poorly understood. W-Beijing Mtb strains are highly prevalent throughout the world and associated with increased drug resistance2. In the early 1990s, closely related MDR W-Beijing Mtb strains (W strains) were identified in large institutional outbreaks in New York City and caused high mortality rates3. The production of interleukin-1ß (IL-1ß) by macrophages coincides with the shift towards aerobic glycolysis, a metabolic process that mediates protection against drug-susceptible Mtb4. Here, using a collection of MDR W-Mtb strains, we demonstrate that the overexpression of Mtb cell wall lipids, phthiocerol dimycocerosates, bypasses the interleukin 1 receptor, type I (IL-1R1) signalling pathway, instead driving the induction of interferon-ß (IFN-ß) to reprogram macrophage metabolism. Importantly, Mtb carrying a drug resistance-conferring single nucleotide polymorphism in rpoB (H445Y)5 can modulate host macrophage metabolic reprogramming. These findings transform our mechanistic understanding of how emerging MDR Mtb strains may acquire drug resistance single nucleotide polymorphisms, thereby altering Mtb surface lipid expression and modulating host macrophage metabolic reprogramming.

PPD-induced monocyte mitochondrial damage is associated with a protective effect to develop tuberculosis in BCG vaccinated individuals: A cohort study.

INTRODUCTION: The mechanisms of mononuclear phagocyte death have been associated with the permissiveness and resistance to mycobacterial replication, but it remains unknown whether or not they help predict the risk of developing TB. OBJECTIVE: To describe the factors associated with the induction of monocyte mitochondrial and membrane damage in response to PPD as well as determine if this type of damage might predict the susceptibility of developing active tuberculosis in a cohort of household contacts (HHCs) from Medellin, Colombia from 2005 to 2008. METHODS: The prospective cohort study contains 2060 HHCs patients with pulmonary tuberculosis who were meticulously followed for two years. A survey of the socio-demographic, clinical, epidemiological factors and blood samples were collected. Mononuclear cell cultures were stimulated with or without PPD and the type of monocyte death was determined by the flow of cytometry, an indicator was also used for its analysis. Logistic regression was adjusted by the Generalized Estimations Equations and the survival was estimated with the Kaplan-Meier and Cox regression. Confidence intervals were used for estimating the association. RESULTS: 1,859 out of 2,060 blood samples of the HHCs patients analyzed showed monocyte death. In response to PPD, 83.4% underwent mitochondrial damage while 50.9% had membrane damage. The membrane damage in response to PPD was higher in children under 4 years (OR: 1.57; (95% CI: 1.1 to 2.4) and the HHCs who slept regularly in the same household has an index case of (OR: 1.54; 95% CI: 1.0 to 2.3). After adjustment by age, comorbidities, nutritional status, proximity to index case and overcrowding, the risk of developing active TB among BCG vaccinated HHCs individuals with induction of mitochondrial damage was HR = 0.19 (95% CI: 0.1 to 0.5). CONCLUSIONS: The induction of monocytes mitochondrial damage by PPD stimulation correlates with protection of TB disease development in BCG-vaccinated HHCs. This represents a potential tool to predict susceptibility of developing active disease in this population.

The role of CD30 and CD153 (CD30L) in the anti-mycobacterial immune response.

The establishment of a protective T-cell response against mycobacterial infections involves different co-stimulatory molecules and their respective ligands. Among these molecules the Tumor Necrosis Factor Receptor Super-family (TNFRSF) and the Tumor Necrosis Factor Super-family (TNFSF) are known to be important members. This review analyzes the evidence that CD30 and CD153 (CD30L), members of the TNFRSF and TNSF, play key roles in the T cell-dependent anti-mycobacterial immune response. Mice deficient in either CD30 or CD153, or treated with antibodies blocking the effects or CD30 and CD153, and infected with M.avium or M.bovis BCG exhibit higher bacterial burden, abnormal inflammatory responses with decreased Th1 responses, this is evidenced by the reduced number of IFN-γ-producing cells. Recent evidence also showed that CD30+ CD153+ Tγδ cells participate in the early stages of M.bovis BCG infection by producing IL-17A. In humans, stimulation of T-cells with mycobacterial antigens induces CD30 expression mainly by CD4+ cells; CD30+ cells have been demonstrated in tissues of patients with tuberculosis (TB) and in positive tuberculin skin test reactions. In addition, the levels of soluble CD30 are increased in serum and BAL of TB patients and these levels seems to correlate with the severity of the disease. These findings suggest that CD30/CD153 interactions during the anti-mycobacterial immune response are important for the establishment and maintenance of a protective response. Further studies would be required to determine whether these molecules may be good clinical biomarkers or potential targets for immune manipulation.

Functional profile of CD4+ and CD8+ T cells in latently infected individuals and patients with active TB.

Tuberculosis (TB) is one of the most important infectious diseases around the world. Several studies have focused on the identification of correlates of protection against TB. Most of them have concentrated on the study of IFN-γ due to its robust association with protection against TB. However, given the complexity of the immune response elicited after Mtb infection, other cytokines should also be considered. In the present study, we evaluated Th1 and Th17 responses and their association with the protection or development of active disease. Therefore, non infected individuals (nonTBi), latently infected individuals (LTBi) and patients with active TB (ATB) were studied. The evaluation of the number of cytokine producing cells by ELISPOT showed a higher number of IFN-γ-producing cells in ATB patients, but no differences were found regarding the number of IL-17 producing cells among studied groups. The evaluation of IFN-γ, IL-2, TNF-α and IL-17 producing CD4+ and CD8+ T cells at 1 day and 6 days of stimulation with mycobacterial antigens suggests the presence of functional signatures associated with latency or active TB. The results presented herein suggest the possible use of the evaluation of Th1-type cytokines, such as IFN-γ and/or TNF-α, as a correlate of protection against TB; however, these results need to be validated for other groups.

Reduced frequency of memory T cells and increased Th17 responses in patients with active tuberculosis.

Phenotypic and functional alterations in Mycobacterium tuberculosis T cell subsets have been reported in patients with active tuberculosis. A better understanding of these alterations will increase the knowledge about immunopathogenesis and also may contribute to the development of new diagnostics and prophylactic strategies. Here, the ex vivo phenotype of CD4(+) and CD8(+) T cells and the frequency and phenotype of gamma interferon (IFN-γ)- and interleukin 17 (IL-17)-producing cells elicited in short-term and long-term cultures following CFP-10 and purified protein derivative (PPD) stimulation were determined in noninfected persons (non-TBi), latently infected persons (LTBi), and patients with active tuberculosis (ATB). Phenotypic characterization of T cells was done based on the expression of CD45RO and CD27. Results show that ATB had a reduced frequency of circulating CD4(+) CD45RO(+) CD27(+) T cells and an increased frequency of CD4(+) CD45RO(-) CD27(+) T cells. ATB also had a higher frequency of circulating IL-17-producing CD4(+) T cells than did LTBi after PPD stimulation, whereas LTBi had more IFN-γ-producing CD4(+) T cells than did non-TBi. The phenotype of IFN-γ-producing cells at 24 h differs from the phenotype of IL-17-producing cells with no differences between LTBi and ATB. At 144 h, IFN-γ- and IL-17-producing cells were mainly CD45RO(+) CD27(+) T cells and they were more frequent in ATB. These results suggest that M. tuberculosis infection induces alterations in T cells which interfere with an adequate specific immune response.

Characterization of CD4 and CD8 T cells producing IFN-γ in human latent and active tuberculosis.

Patients with pulmonary tuberculosis (PTB) frequently have reduced IFN-γ production in response to mycobacterial antigens, compared to individuals with latent Mycobacterium tuberculosis infection (LTBi). However, it is not clear whether this reduced responsiveness is restricted to a particular T cell subset. Herein, PBMCs from 26 PTB patients, 30 household contacts (HHCs) of PTB, and 30 tuberculin positive (TST+) healthy subjects not recently exposed to PTB, were stained with CFSE and stimulated non-specific (PPD) for 120 h, and specific (CFP-10/ESAT-6) and latency (HSpX) mycobacterial antigens for 144 h and the percentage of CD4(+) and CD8(+)IFN-γ(+) T cells responding determined by flow cytometry, in addition to their memory phenotype by the CD45RO and CD27 expression. PTB had decreased frequency of both CD4(+) and CD8(+) precursor cells, as well as decreased number of CD4(+)IFN-γ(+) cells in response to all antigens, whereas CD8(+)IFN-γ(+) cells were decreased in response to PPD and ESAT-6, but not to CFP-10 and HSpX. HHCs exhibited the highest precursor frequencies and IFN-γ responses, irrespective of the antigen employed. The CD4(+)/CD8(+) cell ratios showed that in response to PPD CD4(+) precursor and IFN-γ-producer cells are more frequent than their CD8(+) counterparts, and that PTB have a decreased CD4(+)IFN-γ(+)/CD8(+)IFN-γ(+) ratio in response to PPD, CFP-10, and ESAT-6. CD4(+)IFN-γ(+) and CD8(+)IFN-γ(+) cells exhibited a central memory phenotype (CD45RO(+)CD27(+)), irrespective of the group of subjects and the antigen used for stimulation. In conclusion, PTB patients had a decreased percentage of CD4(+) and CD8(+) precursor cells and CD4(+)IFN-γ(+). HHCs exhibited the highest frequency of CD4(+) and CD8(+) precursors and CD4(+)IFN-γ(+)-producing cells.