Persistence of a Skewed Repertoire of NK Cells in People with HIV-1 on Long-Term Antiretroviral Therapy.

HIV-1 infection greatly alters the NK cell phenotypic and functional repertoire. This is highlighted by the expansion of a rare population of FcRγ- NK cells exhibiting characteristics of traditional immunologic memory in people with HIV (PWH). Although current antiretroviral therapy (ART) effectively controls HIV-1 viremia and disease progression, its impact on HIV-1-associated NK cell abnormalities remains unclear. To address this, we performed a longitudinal analysis detailing conventional and memory-like NK cell characteristics in n = 60 PWH during the first 4 y of ART. Throughout this regimen, a skewed repertoire of cytokine unresponsive FcRγ- memory-like NK cells persisted and accompanied an overall increase in NK surface expression of CD57 and KLRG1, suggestive of progression toward immune senescence. These traits were linked to elevated serum inflammatory biomarkers and increasing Ab titers to human CMV, with human CMV viremia detected in approximately one-third of PWH at years 1-4 of ART. Interestingly, 40% of PWH displayed atypical NK cell subsets, representing intermediate stages of NK-poiesis based on single-cell multiomic trajectory analysis. Our findings indicate that NK cell irregularities persist in PWH despite long-term ART, underscoring the need to better understand the causative mechanisms that prevent full restoration of immune health in PWH.

The Frequency and Function of NKG2C+CD57+ Adaptive NK Cells in Cytomagalovirus Co-Infected People Living with HIV Decline with Duration of Antiretroviral Therapy.

Human cytomegalovirus (CMV) infection drives the expansion and differentiation of natural killer (NK) cells with adaptive-like features. We investigated whether age and time on antiretroviral therapy (ART) influenced adaptive NK cell frequency and functionality. Flow cytometry was used to evaluate the frequency of adaptive and conventional NK cells in 229 CMV+ individuals of whom 170 were people living with HIV (PLWH). The frequency of these NK cell populations producing CD107a, CCL4, IFN-γ or TNF-α was determined following a 6-h antibody dependent (AD) stimulation. Though ART duration and age were correlated, longer time on ART was associated with a reduced frequency of adaptive NK cells. In general, the frequency and functionality of NK cells following AD stimulation did not differ significantly between treated CMV+PLWH and CMV+HIV- persons, suggesting that HIV infection, per se, did not compromise AD NK cell function. AD activation of adaptive NK cells from CMV+PLWH induced lower frequencies of IFN-γ or TNF-α secreting cells in older persons, when compared with younger persons.

Single-cell analysis reveals clonally expanded tumor-associated CD57+ CD8 T cells are enriched in the periphery of patients with metastatic urothelial cancer responding to PD-L1 blockade.

BACKGROUND: A growing body of evidence suggests that T-cell responses against neoantigens are critical regulators of response to immune checkpoint blockade. We previously showed that circulating neoantigen-specific CD8 T cells in patients with lung cancer responding to anti-Programmed death-ligand 1 (PD-L1) (atezolizumab) exhibit a unique phenotype with high expression of CD57, CD244, and KLRG1. Here, we extended our analysis on neoantigen-specific CD8 T cells to patients with metastatic urothelial cancer (mUC) and further profiled total CD8 T cells to identify blood-based predictive biomarkers of response to atezolizumab. METHODS: We identified tumor neoantigens from 20 patients with mUC and profiled their peripheral CD8 T cells using highly multiplexed combinatorial tetramer staining. Another set of patients with mUC treated with atezolizumab (n=30) or chemotherapy (n=40) were selected to profile peripheral CD8 T cells by mass cytometry. Using single-cell transcriptional analysis (single-cell RNA sequencing (scRNA-seq)), together with CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) and paired T-cell receptor (TCR) sequencing, we further characterized peripheral CD8 T cells in a subset of patients (n=16). RESULTS: High frequency of CD57 was observed in neoantigen-specific CD8 T cells in patients with mUC responding to atezolizumab. Extending these findings to bulk CD8 T cells, we found higher frequency of CD57 expressing CD8 T cells before treatment in patients responding to atezolizumab (n=20, p<0.01) but not to chemotherapy. These findings were corroborated in a validation cohort (n=30, p<0.01) and notably were independent of known biomarkers of response. scRNA-seq analysis identified a clonally expanded cluster enriched within CD57+ CD8 T cells in responding patients characterized by higher expression of genes associated with activation, cytotoxicity, and tissue-resident memory markers. Furthermore, compared with CD57- CD8 T cells, TCRs of CD57+ CD8 T cells showed increased overlap with the TCR repertoire of tumor-infiltrating T cells. CONCLUSIONS: Collectively, we show high frequencies of CD57 among neoantigen-specific and bulk CD8 T cells in patients responding to atezolizumab. The TCR repertoire overlap between peripheral CD57+ CD8 T cells and tumor-infiltrating lymphocytes suggest that accumulation of peripheral CD57+ CD8 T cells is reflective of an ongoing antitumor T-cell response. Our findings provide evidence and rationale for using circulating CD8 T cells expressing CD57 as a readily accessible blood-based biomarker for selecting patients with mUC for atezolizumab therapy.

Anti-MAG neuropathy: From biology to clinical management.

The acquired chronic demyelinating neuropathies include a growing number of disease entities that have characteristic, often overlapping, clinical presentations, mediated by distinct immune mechanisms, and responding to different therapies. After the discovery in the early 1980s, that the myelin associated glycoprotein (MAG) is a target antigen in an autoimmune demyelinating neuropathy, assays to measure the presence of anti-MAG antibodies were used as the basis to diagnose the anti-MAG neuropathy. The route was open for describing the clinical characteristics of this new entity as a chronic distal large fiber sensorimotor neuropathy, for studying its pathogenesis and devising specific treatment strategies. The initial use of chemotherapeutic agents was replaced by the introduction in the late 1990s of rituximab, a monoclonal antibody against CD20+ B-cells. Since then, other anti-B cells agents have been introduced. Recently a novel antigen-specific immunotherapy neutralizing the anti-MAG antibodies with a carbohydrate-based ligand mimicking the natural HNK-1 glycoepitope has been described.

The Adaptive Immune Landscape of the Colorectal Adenoma-Carcinoma Sequence.

BACKGROUND: The tumor immune microenvironment exerts a pivotal influence in tumor initiation and progression. The aim of this study was to analyze the immune context of sporadic and familial adenomatous polyposis (FAP) lesions along the colorectal adenoma-carcinoma sequence (ACS). METHODS: We analyzed immune cell counts (CD3+, CD4+, CD8+, Foxp3+, and CD57+), tumor mutation burden (TMB), MHC-I expression and PD-L1 expression of 59 FAP and 74 sporadic colorectal lesions, encompassing adenomas with low-grade dysplasia (LGD) (30 FAP; 30 sporadic), adenomas with high-grade dysplasia (22 FAP; 30 sporadic), and invasive adenocarcinomas (7 FAP; 14 sporadic). RESULTS: The sporadic colorectal ACS was characterized by (1) a stepwise decrease in immune cell counts, (2) an increase in TMB and MHC-I expression, and (3) a lower PD-L1 expression. In FAP lesions, we observed the same patterns, except for an increase in TMB along the ACS. FAP LGD lesions harbored lower Foxp3+ T cell counts than sporadic LGD lesions. A decrease in PD-L1 expression occurred earlier in FAP lesions compared to sporadic ones. CONCLUSIONS: The colorectal ACS is characterized by a progressive loss of adaptive immune infiltrate and by the establishment of a progressively immune cold microenvironment. These changes do not appear to be related with the loss of immunogenicity of tumor cells, or to the onset of an immunosuppressive tumor microenvironment.

Association of Premature Immune Aging and Cytomegalovirus After Solid Organ Transplant.

Immune function is altered with increasing age. Infection with cytomegalovirus (CMV) accelerates age-related immunological changes resulting in expanded oligoclonal memory CD8 T cell populations with impaired proliferation, signaling, and cytokine production. As a consequence, elderly CMV seropositive (CMV+) individuals have increased mortality and impaired responses to other infections in comparison to seronegative (CMV-) individuals of the same age. CMV is also a significant complication after organ transplantation, and recent studies have shown that CMV-associated expansion of memory T cells is accelerated after transplantation. Thus, we investigated whether immune aging is accelerated post-transplant, using a combination of telomere length, flow cytometry phenotyping, and single cell RNA sequencing. Telomere length decreased slightly in the first year after transplantation in a subset of both CMV+ and CMV- recipients with a strong concordance between CD57+ cells and short telomeres. Phenotypically aged cells increased post-transplant specifically in CMV+ recipients, and clonally expanded T cells were enriched for terminally differentiated cells post-transplant. Overall, these findings demonstrate a pattern of accelerated aging of the CD8 T cell compartment in CMV+ transplant recipients.

CMV exposure drives long-term CD57+ CD4 memory T-cell inflation following allogeneic stem cell transplant.

Donor and recipient cytomegalovirus (CMV) serostatus correlate with transplant-related mortality that is associated with reduced survival following allogeneic stem cell transplant (SCT). Prior epidemiologic studies have suggested that CMV seronegative recipients (R-) receiving a CMV-seropositive graft (D+) experience inferior outcomes compared with other serostatus combinations, an observation that appears independent of viral reactivation. We therefore investigated the hypothesis that prior donor CMV exposure irreversibly modifies immunologic function after SCT. We identified a CD4+/CD57+/CD27- T-cell subset that was differentially expressed between D+ and D- transplants and validated results with 120 patient samples. This T-cell subset represents an average of 2.9% (D-/R-), 18% (D-/R+), 12% (D+/R-), and 19.6% (D+/R+) (P < .0001) of the total CD4+ T-cell compartment and stably persists for at least several years post-SCT. Even in the absence of CMV reactivation post-SCT, D+/R- transplants displayed a significant enrichment of these cells compared with D-/R- transplants (P = .0078). These are effector memory cells (CCR7-/CD45RA+/-) that express T-bet, Eomesodermin, granzyme B, secrete Th1 cytokines, and are enriched in CMV-specific T cells. These cells are associated with decreased T-cell receptor diversity (P < .0001) and reduced proportions of major histocompatibility class (MHC) II expressing classical monocytes (P < .0001), myeloid (P = .024), and plasmacytoid dendritic cells (P = .0014). These data describe a highly expanded CD4+ T-cell population and putative mechanisms by which prior donor or recipient CMV exposure may create a lasting immunologic imprint following SCT, providing a rationale for using D- grafts for R- transplant recipients.

Molecular Signatures of Natural Killer Cells in CMV-Associated Anterior Uveitis, A New Type of CMV-Induced Disease in Immunocompetent Individuals.

Cytomegalovirus (CMV) causes clinical issues primarily in immune-suppressed conditions. CMV-associated anterior uveitis (CMV-AU) is a notable new disease entity manifesting recurrent ocular inflammation in immunocompetent individuals. As patient demographics indicated contributions from genetic background and immunosenescence as possible underlying pathological mechanisms, we analyzed the immunogenetics of the cohort in conjunction with cell phenotypes to identify molecular signatures of CMV-AU. Among the immune cell types, natural killer (NK) cells are main responders against CMV. Therefore, we first characterized variants of polymorphic genes that encode differences in CMV-related human NK cell responses (Killer cell Immunoglobulin-like Receptors (KIR) and HLA class I) in 122 CMV-AU patients. The cases were then stratified according to their genetic features and NK cells were analyzed for human CMV-related markers (CD57, KLRG1, NKG2C) by flow cytometry. KIR3DL1 and HLA class I combinations encoding strong receptor-ligand interactions were present at substantially higher frequencies in CMV-AU. In these cases, NK cell profiling revealed expansion of the subset co-expressing CD57 and KLRG1, and together with KIR3DL1 and the CMV-recognizing NKG2C receptor. The findings imply that a mechanism of CMV-AU pathogenesis likely involves CMV-responding NK cells co-expressing CD57/KLRG1/NKG2C that develop on a genetic background of KIR3DL1/HLA-B allotypes encoding strong receptor-ligand interactions.

Increased Frequency of Dysfunctional Siglec-7-CD57+PD-1+ Natural Killer Cells in Patients With Non-alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is a progressive disorder that can develop into liver fibrosis and hepatocellular carcinoma. Natural killer (NK) cells have been shown to protect against liver fibrosis and tumorigenesis, suggesting that they may also play a role in the pathogenesis of NAFLD. Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of inhibitory and activating receptors expressed by many cell types, including NK cells. Here, we investigated the phenotypic profiles of peripheral blood and intrahepatic NK cells, including expression of Siglecs and immune checkpoint molecules, and their association with NK cell function in patients with NAFLD. Immune cells in the peripheral blood of 42 patients with biopsy-proven NAFLD and 13 healthy volunteers (HVs) were identified by mass cytometry. The function of various NK cell subpopulations was assessed by flow cytometric detection of intracellular IFN-γ and CD107a/LAMP-1, a degranulation marker, after in vitro stimulation. We found that peripheral blood from NAFLD patients, regardless of fibrosis stage, contained significantly fewer total CD56+ NK cell and CD56dim NK cell populations compared with HVs, and the CD56dim cells from NAFLD patients were functionally impaired. Among the Siglecs examined, NK cells predominantly expressed Siglec-7 and Siglec-9, and both the expression levels of Siglec-7 and Siglec-9 on NK cells and the frequencies of Siglec-7+CD56dim NK cells were reduced in NAFLD patients. Notably, Siglec-7 levels on CD56dim NK cells were inversely correlated with PD-1, CD57, and ILT2 levels and positively correlated with NKp30 and NKp46 levels. Further subtyping of NK cells identified a highly dysfunctional Siglec-7-CD57+PD-1+CD56dim NK cell subset that was increased in patients with NAFLD, even those with mild liver fibrosis. Intrahepatic NK cells from NAFLD patients expressed elevated levels of NKG2D and CD69, suggesting a more activated phenotype than normal liver NK cells. These data identify a close association between NK cell function and expression of Siglec-7, CD57, and PD-1 that could potentially be therapeutically targeted in NAFLD.

Th22 response induced by Mycobacterium tuberculosis strains is closely related to severity of pulmonary lesions and bacillary load in patients with multi-drug-resistant tuberculosis.

The role of interleukin-22 (IL-22) in the pathogenesis or tissue repair in human tuberculosis (TB) remains to be established. Here, we aimed to explore the ex-vivo and in-vitro T helper 22 (Th22) response in TB patients and healthy donors (HD) induced by different local multi-drug-resistant (MDR) Mvcobacterium tuberculosis (Mtb) strains. For this purpose, peripheral blood mononuclear cells from drug-susceptible (S-TB) MDR-TB patients and HD were stimulated with local MDR strains and the laboratory strain H37Rv. IL-22 and IL-17 expression and senescent status were assessed in CD4+ and CD8+ cells by flow cytometry, while IL-22 amount was measured in plasma and culture supernatants by enzyme-linked immunosorbent assay (ELISA). We found lower IL-22 amounts in plasma from TB patients than HD, together with a decrease in the number of circulating T cells expressing IL-22. In a similar manner, all Mtb strains enhanced IL-22 secretion and expanded IL-22+ cells within CD4+ and CD8+ subsets, being the highest levels detected in S-TB patients. In MDR-TB, low systemic and Mtb-induced Th22 responses associated with high sputum bacillary load and bilateralism of lung lesions, suggesting that Th22 response could be influencing the ability of MDR-TB patients to control bacillary growth and tissue damage. In addition, in MDR-TB patients we observed that the higher the percentage of IL-22+ cells, the lower the proportion of programmed cell death 1 (PD-1)+ or CD57+ T cells. Furthermore, the highest proportion of senescent T cells was associated with severe lung lesions and bacillary load. Thus, T cell senescence would markedly influence Th22 response mounted by MDR-TB patients.

Mass Cytometry Analysis of the NK Cell Receptor-Ligand Repertoire Reveals Unique Differences between Dengue-Infected Children and Adults.

Dengue virus (DENV) is a significant cause of morbidity in many regions of the world, with children at the greatest risk of developing severe dengue. NK cells, characterized by their ability to rapidly recognize and kill virally infected cells, are activated during acute DENV infection. However, their role in viral clearance versus pathogenesis has not been fully elucidated. Our goal was to profile the NK cell receptor-ligand repertoire to provide further insight into the function of NK cells during pediatric and adult DENV infection. We used mass cytometry to phenotype isolate NK cells and PBMCs from a cohort of DENV-infected children and adults. Using unsupervised clustering, we found that pediatric DENV infection leads to a decrease in total NK cell frequency with a reduction in the percentage of CD56dimCD38bright NK cells and an increase in the percentage of CD56dimperforinbright NK cells. No such changes were observed in adults. Next, we identified markers predictive of DENV infection using a differential state test. In adults, NK cell expression of activation markers, including CD69, perforin, and Fas-L, and myeloid cell expression of activating NK cell ligands, namely Fas, were predictive of infection. In contrast, increased NK cell expression of the maturation marker CD57 and myeloid cell expression of inhibitory ligands, such as HLA class I molecules, were predictive of pediatric DENV infection. These findings suggest that acute pediatric DENV infection may result in diminished NK cell activation, which could contribute to enhanced pathogenesis and disease severity.

The Oncometabolite 5'-Deoxy-5'-Methylthioadenosine Blocks Multiple Signaling Pathways of NK Cell Activation.

Tumor cells develop various mechanisms to escape immune surveillance. In this context, oncometabolites secreted by tumor cells due to deregulated metabolic pathways, have been in the spotlight of researchers during the last years. 5'-Deoxy-5'-methylthioadenosine (MTA) phosphorylase (MTAP) deficiency in tumors results in the accumulation of MTA within the tumor microenvironment and thereby negatively influencing immune functions of various immune cells, including T and NK cells. The influence of MTA on T cell activation has been recently described in more detail, while its impact on NK cells is still largely unknown. Therefore, we aimed to illuminate the molecular mechanism of MTA-induced NK cell dysfunction. NK cell cytotoxicity against target cells was reduced in the presence of MTA in a dose-dependent manner, while NK cell viability remained unaffected. Furthermore, we revealed that MTA blocks NK cell degranulation and cytokine production upon target cell engagement as well as upon antibody stimulation. Interestingly, the immune-suppressive effect of MTA was less pronounced in healthy donors harboring an expansion of NKG2C+ NK cells. Finally, we demonstrated that MTA interferes with various signaling pathways downstream of the CD16 receptor upon NK cell activation, including the PI3K/AKT/S6, MAPK/ERK, and NF-κB pathways. In summary, we revealed that MTA blocks NK cell functions like cytotoxicity and cytokine production by interfering with the signaling cascade of activating NK cell receptors. Specific targeting of MTA metabolism in MTAP-deficient tumors therefore could offer a promising new strategy to reverse immune dysfunction of NK cells within the tumor microenvironment.

NK Cell Activity and CD57+/NKG2Chigh Phenotype Are Increased in Men Who Have Sex With Men at High Risk for HIV.

Introduction: The HIV-exposed seronegative (HESN) status is for individuals who remain seronegative despite repeated exposure to HIV. One of the main cohorts within this group is men who have sex with men (MSM). Studies of this cohort have revealed different immunological and genetic mechanisms that can explain the phenomenon of natural HIV resistance. NK cells' higher effector capacity is related to natural resistance to HIV. Besides, a new population of NK cells with adaptive features was described recently. These cells are increased in some HESN cohorts and appear to be involved in better control of viral replication in primarily HIV-infected subjects. The present study evaluated the role of NK cells in the natural resistance to HIV-1 infection in MSM. Methodology: Phenotypic and functional features were evaluated in NK cells from two groups of MSM, at different risks of HIV infection, according to the number of sexual partners. The production of IFN-γ and ß-chemokines was included in the analysis, as well as the cytotoxic capacity and adaptive NK cell frequency. Genetic features, such as HLA and KIR allele frequencies, were also explored. Results: High-risk MSM exhibit an increased frequency of fully mature and CD57+/NKG2Chigh NK cells. These individuals also show higher cytotoxic capacity and IFN-γ production in response to K562 stimuli. NK cells with a CD107a+/IFN-γ+ functional profile were found more frequently and displayed higher IFN-γ production capacity among high-risk MSM than among low-risk MSM. The protective allele HLA-B∗18 was only present in the high-risk MSM group as well as HLA-B∗ 39. The protective phenotype KIR3DL1/S1-HLA-B∗Bw4, in a homozygous state, was particularly abundant in the high-risk population. Notably, some of these functional features were related to higher frequencies of mature and CD57+/NKG2Chigh NK cells, which, in turn, were associated with a higher number of sexual partners. Conclusion: The changes observed in the NK cell compartment can be driven by the magnitude of sexual exposure and immunological challenges of high-risk individuals, which could influence their resistance/susceptibility to HIV infection.

CD56dim CD57- NKG2C+ NK cells retaining proliferative potential are possible precursors of CD57+ NKG2C+ memory-like NK cells.

Formation of the adaptive-like NK cell subset in response to HCMV infection is associated with epigenetic rearrangements, accompanied by multiple changes in the protein expression. This includes a decrease in the expression level of the adapter chain FcεRIγ, NKp30, and NKG2A receptors and an increase in the expression of NKG2C receptor, some KIR family receptors, and co-stimulating molecule CD2. Besides, adaptive-like NK cells are characterized by surface expression of CD57, a marker of highly differentiated cells. Here, it is shown that CD57-negative CD56dim NKG2C+ NK cells may undergo the same changes, as established by the similarity of the phenotypic expression pattern with that of the adaptive-like CD57+ NKG2C+ NK cells. Regardless of their differentiation stage, NKG2C-positive NK cells had increased HLA-DR expression indicating an activated state, both ex vivo and after cultivation in stimulating conditions. Additionally, CD57- NKG2C+ NK cells exhibited better proliferative activity compared to CD57+ NKG2C+ and NKG2C- NK cells, while retaining high level of natural cytotoxicity. Thus, CD57- NKG2C+ NK cells may represent a less differentiated, but readily expanding stage of the adaptive-like CD57+ NKG2C+ NK cells. Moreover, it is shown that NK cells have certain phenotypic plasticity and may both lose NKG2C expression and acquire it de novo during proliferation, induced by IL-2 and K562-mbIL21 feeder cells.

Phenotype and molecular signature of CD8+ T cell subsets in T cell- mediated rejections after kidney transplantation.

We investigated the phenotype and molecular signatures of CD8+ T cell subsets in kidney-transplant recipients (KTRs) with biopsy-proven T cell-mediated rejection (TCMR). We included 121 KTRs and divided them into three groups according to the pathologic or clinical diagnosis: Normal biopsy control (NC)(n = 32), TCMR (n = 50), and long-term graft survival (LTGS)(n = 39). We used flowcytometry and microarray to analyze the phenotype and molecular signatures of CD8+ T cell subsets using peripheral blood from those patients and analyzed significant gene expressions according to CD8+ T cell subsets. We investigated whether the analysis of CD8+ T cell subsets is useful for predicting the development of TCMR. CCR7+CD8+ T cells significantly decreased, but CD28nullCD57+CD8+ T cells and CCR7-CD45RA+CD8+ T cells showed an increase in the TCMR group compared to other groups (p<0.05 for each); hence CCR7+CD8+ T cells showed significant negative correlations to both effector CD8+ T cells. We identified genes significantly associated with the change of CCR7+CD8+ T, CCR7-CD45RA+CD8+ T, and CD28nullCD57+CD8+ T cells in an ex vivo study and found that most of them were included in the significant genes on in vitro CCR7+CD8+ T cells. Finally, the decrease of CCR7+CD8+ T cells relative to CD28nullCD57+ T or CCR7-CD45RA+CD8+ T cells can predict TCMR significantly in the whole clinical cohort. In conclusion, phenotype and molecular signature of CD8+ T subsets showed a significant relationship to the development of TCMR; hence monitoring of CD8+ T cell subsets may be a useful for predicting TCMR in KTRs.

Inflammescent CX3CR1+CD57+CD8+ T cells are generated and expanded by IL-15.

HIV infection is associated with an increase in the proportion of activated CD8+ memory T cells (Tmem) that express CX3CR1, but how these cells are generated and maintained in vivo is unclear. We demonstrate that increased CX3CR1 expression on CD8+ Tmem in people living with HIV (PLWH) is dependent on coinfection with human CMV, and CX3CR1+CD8+ Tmem are enriched for a putatively immunosenescent CD57+CD28- phenotype. The cytokine IL-15 promotes the phenotype, survival, and proliferation of CX3CR1+CD57+CD8+ Tmem in vitro, whereas T cell receptor stimulation leads to their death. IL-15-driven survival is dependent on STAT5 and Bcl-2 activity, and IL-15-induced proliferation requires STAT5 and mTORC1. Thus, we identify mechanistic pathways that could explain how "inflammescent" CX3CR1+CD57+ CD8+ Tmem dominate the overall memory T cell pool in CMV-seropositive PLWH and that support reevaluation of immune senescence as a nonproliferative dead end.

Ageing dangerously; homing of senescent CD8 T cells in cutaneous Leishmaniasis.

Both CD8+ T cells and NK cells contribute to the immune response against the protozoan Leishmania parasite. Both are able to generate IFN-γ and both display cytotoxic features. These features may enable them to not only contribute to parasite clearance but also to cause immune-mediated pathology. This pathology is evident, for example, in the Leismania-induced skin lesions found in patients with cutaneous leismaniasis (CL). Here we highlight new data demonstrating that CD8+ T cells and NK cells in CL display a highly cytotoxic senescent phenotype, and that the senescent T cells play a major role in mediating skin pathology. This is the first demonstration that senescent CD8 T cells contribute to immunopathology in vivo.

Compartmentalized cytotoxic immune response leads to distinct pathogenic roles of natural killer and senescent CD8+ T cells in human cutaneous leishmaniasis.

Cytotoxic activity mediated by CD8+ T cells is the main signature of the immunopathogenesis of cutaneous leishmaniasis (CL). Here, we performed a broad evaluation of natural killer (NK) cell phenotypic and functional features during cutaneous leishmaniasis. We demonstrate for the first time that CL patients present the accumulation of circulating NK cells with multiple features of replicative senescence including low proliferative capacity and shorter telomeres, elevated expression of CD57, KLRG1 but diminished CD27 stimulatory receptor expression. Moreover, they exhibited higher cytotoxic and inflammatory potential than age-matched controls. The accumulation of circulating senescent NK cells (CD56dim  CD57bright ) correlated positively with skin lesion size in the same patients, suggesting that they, like circulating senescent CD8+ T cells, may contribute to the immunopathology of CL. However, this senescent population had lower cutaneous lymphocyte antigen expression and so had diminished skin-homing potential compared with total or senescent CD8+ T cells. This was confirmed in CL skin lesions where we found a predominance of CD8+ T cells (both senescent and non-senescent) that correlated with the severity of the disease. Although there was also a correlation between the proportions of senescent NK cells (CD56+  CD57+ ) in the skin and lesion size, this was less evident. Collectively our results demonstrate first-hand that senescent cytotoxic cells may mediate skin pathology during human cutaneous leishmaniasis. However, as senescent cytotoxic CD8+ T cells predominate in the skin lesions, they may have a greater role than NK cells in mediating the non-specific skin damage in CL.

Causes of double-negative T-cell lymphocytosis in children and adults.

AIMS: The causes and diagnosis of 'double-negative' (CD3+CD4-CD8-) T-cell lymphocytosis are not well studied. We aimed to define the causes of double-negative T-cell lymphocytosis in children and adults, and to identify simple clinical and laboratory features that would help to differentiate between the underlying conditions. METHODS: We collected clinical and laboratory data on 10 children and 30 adults with significantly increased peripheral-blood double-negative T-cells (>10% of total lymphocytes). We identified conditions associated with double-negative T-lymphocytosis with flow cytometry, peripheral-blood morphology and T-cell receptor-gene rearrangement studies. Patients were assigned to diagnostic categories on the basis of these test results. RESULTS AND CONCLUSIONS: The causes of double-negative T-cell lymphocytosis in children were autoimmune lymphoproliferative syndrome (ALPS) and reactive γ/δ Τ-lymphocytosis. T-cell large granular lymphocyte (T-LGL) leukaemia, reactive γ/δ T-lymphocytosis and hepatosplenic T-cell lymphoma (HSTL) were the the most common disorders underlying double-negative T-cell lymphocytosis in adults. Less common causes included hypereosinophilic syndrome, peripheral T-cell lymphoma, ALPS and monoclonal, double-negative T-lymphocytosis of uncertain significance. CD5/CD7/Vδ2 expression and absolute double-negative lymphocyte count (<1.8×109/L) were useful discriminators for distinguishing patients with reactive γ/δ T-lymphocytosis from those with γ/δ lymphoproliferative disorders. Differentiating between γ/δ T-LGL and HSTL can be difficult. Expression of CD57 and cellular morphology (pale cytoplasm with distinct granules) would support a diagnosis of γ/δ T-LGL.

CD28 and CD57 define four populations with distinct phenotypic properties within human CD8+ T cells.

After repeated antigen exposure, both memory and terminally differentiated cells can be generated within CD8+ T cells. Although, during their differentiation, activated CD8+ T cells may first lose CD28, and CD28- cells may eventually express CD57 as a subsequent step, a population of CD28+ CD57+ (DP) CD8+ T cells can be identified in the peripheral blood. How this population is distinct from CD28- CD57- (DN) CD8+ T cells, and from the better characterized non-activated/early-activated CD28+ CD57- and senescent-like CD28- CD57+ CD8+ T cell subsets is currently unknown. Here, RNA expression of the four CD8+ T cell subsets isolated from human PBMCs was analyzed using microarrays. DN cells were more similar to "early" highly differentiated cells, with decreased TNF and IFN-γ production, impaired DNA damage response and apoptosis. Conversely, increased apoptosis and expression of cytokines, co-inhibitory, and chemokine receptors were found in DP cells. Higher levels of DP CD8+ T cells were observed 7 days after Hepatitis B vaccination, and decreased levels of DP cells were found in rheumatoid arthritis patients. More DP and DN CD8+ T cells were present in the bone marrow, in comparison with PBMCs. In summary, our results indicate that DP and DN cells are distinct CD8+ T cell subsets displaying defined properties.