Killer cell immunoglobulin-like receptor (KIR) genes and their HLA ligands in a Brazilian population.

Killer cell immunoglobulin-like receptors (KIR) exhibit extensive diversity, giving rise to different KIR profiles in populations worldwide. This study aimed to investigate the distribution of KIR genes and HLA ligands in a population from Campinas, southeastern Brazil (n = 292), and to compare their results with other populations. A comprehensive analysis of population-specific genes, genotype, and haplotype frequencies of KIR may facilitate a better understanding of their evolution and role in immunity. The genotyping of 16 KIR genes and HLA class I alleles was performed by the reverse sequence-specific oligonucleotide methodology using the Luminex platform (One Lambda, Inc., Canoga Park, CA). The framework genes were present in all individuals, with the most common non-framework KIR genes detected being KIR2DP1(96.6%), KIR2DL1(95.5%), KIR3DL1(94.5%), KIR2DS4(93.8%) and KIR2DL3(87.3%). KIR2DS1, KIR2DS3, KIR2DS5, and KIR3DS1 presented frequencies below 40%. KIR2DL2, KIR2DL5, and KIR2DS2 showed intermediate frequencies (between53% and 58%). The activating gene KIR2DS5 was the least common in this population (30.8%). Forty-five KIR profiles were found with the commonest being the homozygous A haplotype (27.4%). The distribution of KIR genes in the Brazilian population is similar to Caucasian European and Euro-descendant populations.

Uncovering the significance of expanded CD8+ large granular lymphocytes in inclusion body myositis: Insights into T cell phenotype and functional alterations, and disease severity.

Introduction: Inclusion body myositis (IBM) is a progressive inflammatory myopathy characterised by skeletal muscle infiltration and myofibre invasion by CD8+ T lymphocytes. In some cases, IBM has been reported to be associated with a systemic lymphoproliferative disorder of CD8+ T cells exhibiting a highly differentiated effector phenotype known as T cell Large Granular Lymphocytic Leukemia (T-LGLL). Methods: We investigated the incidence of a CD8+ T-LGL lymphoproliferative disorder in 85 IBM patients and an aged-matched group of 56 Healthy Controls (HC). Further, we analysed the phenotypical characteristics of the expanded T-LGLs and investigated whether their occurrence was associated with any particular HLA alleles or clinical characteristics. Results: Blood cell analysis by flow cytometry revealed expansion of T-LGLs in 34 of the 85 (40%) IBM patients. The T cell immunophenotype of T-LGLHIGH patients was characterised by increased expression of surface molecules including CD57 and KLRG1, and to a lesser extent of CD94 and CD56 predominantly in CD8+ T cells, although we also observed modest changes in CD4+ T cells and γδ T cells. Analysis of Ki67 in CD57+ KLRG1+ T cells revealed that only a small proportion of these cells was proliferating. Comparative analysis of CD8+ and CD4+ T cells isolated from matched blood and muscle samples donated by three patients indicated a consistent pattern of more pronounced alterations in muscles, although not significant due to small sample size. In the T-LGLHIGH patient group, we found increased frequencies of perforin-producing CD8+ and CD4+ T cells that were moderately correlated to combined CD57 and KLRG1 expression. Investigation of the HLA haplotypes of 75 IBM patients identified that carriage of the HLA-C*14:02:01 allele was significantly higher in T-LGLHIGH compared to T-LGLLOW individuals. Expansion of T-LGL was not significantly associated with seropositivity patient status for anti-cytosolic 5'-nucleotidase 1A autoantibodies. Clinically, the age at disease onset and disease duration were similar in the T-LGLHIGH and T-LGLLOW patient groups. However, metadata analysis of functional alterations indicated that patients with expanded T-LGL more frequently relied on mobility aids than T-LGLLOW patients indicating greater disease severity. Conclusion: Altogether, these results suggest that T-LGL expansion occurring in IBM patients is correlated with exacerbated immune dysregulation and increased disease burden.

Engineering of chimeric natural killer cell receptors to develop precision adoptive immunotherapies for cancer.

Natural killer (NK) cells are innate immune effectors which play a crucial role in recognizing and eliminating virally infected and cancerous cells. They effectively distinguish between healthy and distressed self through the integration of signals delivered by germline-encoded activating and inhibitory cell surface receptors. The frequent up-regulation of stress markers on genetically unstable cancer cells has prompted the development of novel immunotherapies that exploit such innate receptors. One prominent example entails the development of chimeric antigen receptors (CAR) that detect cell surface ligands bound by NK receptors, coupling this engagement to the delivery of tailored immune activating signals. Here, we review strategies to engineer CARs in which specificity is conferred by natural killer group 2D (NKG2D) or other NK receptor types. Multiple preclinical studies have demonstrated the remarkable ability of chimeric NK receptor-targeted T cells and NK cells to effectively and specifically eliminate cancer cells and to reject established tumour burdens. Importantly, such systems act not only acutely but, in some cases, they also incite immunological memory. Moreover, CARs targeted with the NKG2D ligand binding domain have also been shown to disrupt the tumour microenvironment, through the targeting of suppressive T regulatory cells, myeloid-derived suppressor cells and tumour vasculature. Collectively, these findings have led to the initiation of early-phase clinical trials evaluating both autologous and allogeneic NKG2D-targeted CAR T cells in the haematological and solid tumour settings.

Enhancing Natural Killer and CD8+ T Cell-Mediated Anticancer Cytotoxicity and Proliferation of CD8+ T Cells with HLA-E Monospecific Monoclonal Antibodies.

Cytotoxic NK/CD8+ T cells interact with MHC-I ligands on tumor cells through either activating or inhibiting receptors. One of the inhibitory receptors is CD94/NKG2A. The NK/CD8+ T cell cytotoxic capability is lost when tumor-associated human leukocyte antigen, HLA-E, binds the CD94/NKG2A receptor, resulting in tumor progression and reduced survival. Failure of cancer patients to respond to natural killer (NK) cell therapies could be due to HLA-E overexpression in tumor tissues. Preventing the inhibitory receptor-ligand interaction by either receptor- or ligand-specific monoclonal antibodies (mAbs) is an innovative passive immunotherapeutic strategy for cancer. Since receptors and ligands can be monomeric or homo- or heterodimeric proteins, the efficacy of mAbs may rely on their ability to distinguish monospecific (private) functional epitopes from nonfunctional common (public) epitopes. We developed monospecific anti-HLA-E mAbs (e.g., TFL-033) that recognize only HLA-E-specific epitopes, but not epitopes shared with other HLA class-I loci as occurs with currently available polyreactive anti-HLA-E mAbs. Interestingly the amino acid sequences in the α1 and α2 helices of HLA-E, critical for the recognition of the mAb TFL-033, are strikingly the same sequences recognized by the CD94/NKG2A inhibitory receptors on NK/CD8+ cells. Such monospecific mAbs can block the CD94/NKG2A interaction with HLA-E to restore NK cell and CD8+ anticancer cell cytotoxicity. Furthermore, the HLA-E monospecific mAbs significantly promoted the proliferation of the CD4-/CD8+ T cells. These monospecific mAbs are also invaluable for the specific demonstration of HLA-E on tumor biopsies, potentially indicating those tumors most likely to respond to such therapy. Thus, they can be used to enhance passive immunotherapy once phased preclinical studies and clinical trials are completed. On principle, we postulate that NK cell passive immunotherapy should capitalize on both of these features of monospecific HLA-E mAbs, that is, the specific determination HLA-E expression on a particular tumor and the enhancement of NK cell/CD8+ cytotoxicity if HLA-E positive.

Mesenchymal Stromal Cells and Natural Killer Cells: A Complex Story of Love and Hate.

Mesenchymal stromal cells (MSCs), characterized by both multidifferentiation potential and potent immunomodulatory capacity, represent a promising, safe and powerful cell based-therapy for repairing tissue damage and/or treating diseases associated with aberrant immune responses. Natural killer (NK) cells are granular lymphocytes of the innate immune system that function alone or in combination with other immune cells to combat both tumors and virally infected cells. After their infusion, MSCs are guided by host inflammatory elements and can interact with different immune cells, particularly those of the innate immune system. Although some breakthroughs have been achieved in understanding these interactions, much remains to be determined. In this review, we discuss the complex interactions between NK cells and MSCs, particularly the importance of improving the therapeutic value of MSCs.

Non-linear multidimensional flow cytometry analyses delineate NK cell phenotypes in normal and HIV-infected chimpanzees.

Natural killer (NK) cells are primary immune effector cells with both innate and potentially adaptive functions against viral infections, but commonly become exhausted or dysfunctional during chronic diseases such as human immunodeficiency virus (HIV). Chimpanzees are the closest genetic relatives of humans and have been previously used in immunology, behavior and disease models. Due to their similarities to humans, a better understanding of chimpanzee immunology, particularly innate immune cells, can lend insight into the evolution of human immunology, as well as response to disease. However, the phenotype of NK cells has been poorly defined. In order to define NK cell phenotypes, we unbiasedly quantified NK cell markers among mononuclear cells in both naive and HIV-infected chimpanzees by flow cytometry. We identified NKG2D and NKp46 as the most dominant stable NK cells markers using multidimensional data reduction analyses. Other traditional NK cell markers such as CD8α, CD16 and perforin fluctuated during infection, while some such as CD56, NKG2A and NKp30 were generally unaltered by HIV infection, but did not delineate the full NK cell repertoire. Taken together, these data indicate that phenotypic dysregulation may not be pronounced during HIV infection of chimpanzees, but traditional NK cell phenotyping used for both humans and other non-human primate species may need to be revised to accurately identify chimpanzee NK cells.

Phenotype of NK-Like CD8(+) T Cells with Innate Features in Humans and Their Relevance in Cancer Diseases.

Unconventional T cells are defined by their capacity to respond to signals other than the well-known complex of peptides and major histocompatibility complex proteins. Among the burgeoning family of unconventional T cells, innate-like CD8(+) T cells in the mouse were discovered in the early 2000s. This subset of CD8(+) T cells bears a memory phenotype without having encountered a foreign antigen and can respond to innate-like IL-12 + IL-18 stimulation. Although the concept of innate memory CD8(+) T cells is now well established in mice, whether an equivalent memory NK-like T-cell population exists in humans remains under debate. We recently reported that CD8(+) T cells responding to innate-like IL-12 + IL-18 stimulation and co-expressing the transcription factor Eomesodermin (Eomes) and KIR/NKG2A membrane receptors with a memory/EMRA phenotype may represent a new, functionally distinct innate T cell subset in humans. In this review, after a summary on the known innate CD8(+) T-cell features in the mouse, we propose Eomes together with KIR/NKG2A and CD49d as a signature to standardize the identification of this innate CD8(+) T-cell subset in humans. Next, we discuss IL-4 and IL-15 involvement in the generation of innate CD8(+) T cells and particularly its possible dependency on the promyelocytic leukemia zinc-finger factor expressing iNKT cells, an innate T cell subset well documented for its susceptibility to tumor immune subversion. After that, focusing on cancer diseases, we provide new insights into the potential role of these innate CD8(+) T cells in a physiopathological context in humans. Based on empirical data obtained in cases of chronic myeloid leukemia, a myeloproliferative syndrome controlled by the immune system, and in solid tumors, we observe both the possible contribution of innate CD8(+) T cells to cancer disease control and their susceptibility to tumor immune subversion. Finally, we note that during tumor progression, innate CD8(+) T lymphocytes could be controlled by immune checkpoints. This study significantly contributes to understanding of the role of NK-like CD8(+) T cells and raises the question of the possible involvement of an iNKT/innate CD8(+) T cell axis in cancer.

Convergence of Innate and Adaptive Immunity during Human Aging.

Aging is associated with profound changes in the human immune system, a phenomenon referred to as immunosenescence. This complex immune remodeling affects the adaptive immune system and the CD8+ T cell compartment in particular, leading to the accumulation of terminally differentiated T cells, which can rapidly exert their effector functions at the expenses of a limited proliferative potential. In this review, we will discuss evidence suggesting that senescent αßCD8+ T cells acquire the hallmarks of innate-like T cells and use recently acquired NK cell receptors as an alternative mechanism to mediate rapid effector functions. These cells concomitantly lose expression of co-stimulatory receptors and exhibit decreased T cell receptor signaling, suggesting a functional shift away from antigen-specific activation. The convergence of innate and adaptive features in senescent T cells challenges the classic division between innate and adaptive immune systems. Innate-like T cells are particularly important for stress and tumor surveillance, and we propose a new role for these cells in aging, where the acquisition of innate-like functions may represent a beneficial adaptation to an increased burden of malignancy with age, although it may also pose a higher risk of autoimmune disorders.

Underground Adaptation to a Hostile Environment: Acute Myeloid Leukemia vs. Natural Killer Cells.

Acute myeloid leukemia (AML) is a heterogeneous group of malignancies which incidence increases with age. The disease affects the differentiation of hematopoietic stem or precursor cells in the bone marrow and can be related to abnormal cytogenetic and/or specific mutational patterns. AML blasts can be sensitive to natural killer (NK) cell antitumor response. However, NK cells are frequently defective in AML patients leading to tumor escape. NK cell defects affect not only the expression of the activating NK receptors, including the natural cytotoxicity receptors, the NK group 2, member D, and the DNAX accessory molecule-1, but also cytotoxicity and IFN-γ release. Such perturbations in NK cell physiology could be related to the adaptation of the AML to the immune pressure and more generally to patient's clinical features. Various mechanisms are potentially involved in the inhibition of NK-cell functions in AML, including defects in the normal lymphopoiesis, reduced expression of activating receptors through cell-to-cell contacts, and production of immunosuppressive soluble agents by leukemic blasts. Therefore, the continuous cross-talk between AML and NK cells participates to the leukemia immune escape and eventually to patient's relapse. Methods to restore or stimulate NK cells seem to be attractive strategies to treat patients once the complete remission is achieved. Moreover, our capacity in stimulating the NK cell functions could lead to the development of preemptive strategies to eliminate leukemia-initiating cells before the emergence of the disease in elderly individuals presenting preleukemic mutations in hematopoietic stem cells.

Adaptive Memory of Human NK-like CD8+ T-Cells to Aging, and Viral and Tumor Antigens.

Human natural killer (NK)-like CD8+ T-cells are singular T-cells that express both T and NK cell markers such as CD56; their frequencies depend on their differentiation and activation during their lifetime. There is evidence of the presence of these innate CD8+ T-cells in the human umbilical cord, highlighting the necessity of investigating whether the NK-like CD8+ T-cells arise in the early stages of life (gestation). Based on the presence of cell surface markers, these cells have also been referred to as CD8+KIR+ T-cells, innate CD8+ T-cells, CD8+CD28-KIR+ T-cells or NKT-like CD8+CD56+ cells. However, the functional and co-signaling significance of these NK cell receptors on NK-like CD8+ T-cells is less clear. Also, the diverse array of costimulatory and co-inhibitory receptors are spatially and temporally regulated and may have distinct overlapping functions on NK-like CD8+ T-cell priming, activation, differentiation, and memory responses associated with different cell phenotypes. Currently, there is no consensus regarding the functional properties and phenotypic characterization of human NK-like CD8+ T-cells. Environmental factors, such as aging, autoimmunity, inflammation, viral antigen re-exposure, or the presence of persistent tumor antigens have been shown to allow differentiation ("adaptation") of the NK-like CD8+ T-cells; the elucidation of this differentiation process and a greater understanding of the characteristics of these cells could be important for their eventual in potential therapeutic applications aimed at improving protective immunity. This review will attempt to elucidate an understanding of the characteristics of these cells with the goal toward their eventual use in potential therapeutic applications aimed at improving protective immunity.

Divide, Conquer, and Sense: CD8+CD28- T Cells in Perspective.

Understanding the rationale for the generation of a pool of highly differentiated effector memory CD8+ T cells displaying a weakened capacity to scrutinize for peptides complexed with major histocompatibility class I molecules via their T cell receptor, lacking the "signal 2" CD28 receptor, and yet expressing a highly diverse array of innate receptors, from natural killer receptors, interleukin receptors, and damage-associated molecular pattern receptors, among others, is one of the most challenging issues in contemporary human immunology. The prevalence of these differentiated CD8+ T cells, also known as CD8+CD28-, CD8+KIR+, NK-like CD8+ T cells, or innate CD8+ T cells, in non-lymphoid organs and tissues, in peripheral blood of healthy elderly, namely centenarians, but also in stressful and chronic inflammatory conditions suggests that they are not merely end-of-the-line dysfunctional cells. These experienced CD8+ T cells are highly diverse and capable of sensing a variety of TCR-independent signals, which enables them to respond and fine-tune tissue homeostasis.

Distinct and Synergistic Contributions of Epithelial Stress and Adaptive Immunity to Functions of Intraepithelial Killer Cells and Active Celiac Disease.

BACKGROUND & AIMS: The mechanisms of tissue destruction during progression of celiac disease are poorly defined. It is not clear how tissue stress and adaptive immunity contribute to the activation of intraepithelial cytotoxic T cells and the development of villous atrophy. We analyzed epithelial cells and intraepithelial cytotoxic T cells in family members of patients with celiac disease, who were without any signs of adaptive antigluten immunity, and in potential celiac disease patients, who have antibodies against tissue transglutaminase 2 in the absence of villous atrophy. METHODS: We collected blood and intestinal biopsy specimens from 268 patients at tertiary medical centers in the United States and Italy from 2004 to 2012. All subjects had normal small intestinal histology. Study groups included healthy individuals with no family history of celiac disease or antibodies against tissue transglutaminase 2 (controls), healthy family members of patients with celiac disease, and potential celiac disease patients. Intraepithelial cytotoxic T cells were isolated and levels of inhibitory and activating natural killer (NK) cells were measured by flow cytometry. Levels of heat shock protein (HSP) and interleukin 15 were measured by immunohistochemistry, and ultrastructural alterations in intestinal epithelial cells (IECs) were assessed by electron microscopy. RESULTS: IECs from subjects with a family history of celiac disease, but not from subjects who already had immunity to gluten, expressed higher levels of HS27, HSP70, and interleukin-15 than controls; their IECs also had ultrastructural alterations. Intraepithelial cytotoxic T cells from relatives of patients with celiac disease expressed higher levels of activating NK receptors than cells from controls, although at lower levels than patients with active celiac disease, and without loss of inhibitory receptors for NK cells. Intraepithelial cytotoxic T cells from potential celiac disease patients failed to up-regulate activating NK receptors. CONCLUSIONS: A significant subset of healthy family members of patients with celiac disease with normal intestinal architecture had epithelial alterations, detectable by immunohistochemistry and electron microscopy. The adaptive immune response to gluten appears to act in synergy with epithelial stress to allow intraepithelial cytotoxic T cells to kill epithelial cells and induce villous atrophy in patients with active celiac disease.

Five Layers of Receptor Signaling in γδ T-Cell Differentiation and Activation.

The contributions of γδ T-cells to immunity to infection or tumors critically depend on their activation and differentiation into effectors capable of secreting cytokines and killing infected or transformed cells. These processes are molecularly controlled by surface receptors that capture key extracellular cues and convey downstream intracellular signals that regulate γδ T-cell physiology. The understanding of how environmental signals are integrated by γδ T-cells is critical for their manipulation in clinical settings. Here, we discuss how different classes of surface receptors impact on human and murine γδ T-cell differentiation, activation, and expansion. In particular, we review the role of five receptor types: the T-cell receptor (TCR), costimulatory receptors, cytokine receptors, NK receptors, and inhibitory receptors. Some of the key players are the costimulatory receptors CD27 and CD28, which differentially impact on pro-inflammatory subsets of γδ T-cells; the cytokine receptors IL-2R, IL-7R, and IL-15R, which drive functional differentiation and expansion of γδ T-cells; the NK receptor NKG2D and its contribution to γδ T-cell cytotoxicity; and the inhibitory receptors PD-1 and BTLA that control γδ T-cell homeostasis. We discuss these and other receptors in the context of a five-step model of receptor signaling in γδ T-cell differentiation and activation, and discuss its implications for the manipulation of γδ T-cells in immunotherapy.

Involvement of CD161+ Vδ1+ γδ T cells in systemic sclerosis: association with interstitial pneumonia.

OBJECTIVE: Interstitial pneumonia (IP) is a chronic progressive interstitial lung disease associated with high mortality and poor prognosis. However, the pathogenesis of IP remains to be elucidated. The aim of this study was to clarify the role of CD161(+) Vδ1(+) γδ T cells in SSc patients with IP. METHODS: The proportion of CD161(+) Vδ1(+) γδ T cells in peripheral blood mononuclear cells (PBMCs) and serum sialylated carbohydrate antigen (KL-6) levels were determined. GeneChip analysis was performed with CD161(-) and CD161(+) Vδ1(+) γδ T cells. Cytokine and chemokine expression from CD161(+) Vδ1(+) γδ T cells was measured and used to evaluate the effect of culture supernatant on fibroblast proliferation. RESULTS: The proportion of CD161(+) Vδ1(+) γδ T cells was significantly higher in SSc than healthy controls (HCs) and correlated negatively with serum KL-6 levels in IP-positive SSc patients. The gene and mRNA expression level of chemokine ligand 3 (CCL3) was markedly higher in CD161(+) Vδ1(+) γδ T cells than in CD161(-) Vδ1(+) γδ T cells. CD161(+) Vδ1(+) γδ T cells in IP-positive SSc patients showed higher production of CCL3 and lower production of IFN-γ than in HCs. Culture supernatant derived from IP-negative and IP-positive SSc patients promoted fibroblast proliferation, whereas that from HCs did not. CONCLUSION: The small proportion and the altered cell functions of CD161(+) Vδ1(+) γδ T cells among PBMCs in SSc patients play a role in the pathogenesis of IP. These findings suggest that CD161(+) Vδ1(+) γδ T cells may play a regulatory role in the pathogenesis of IP in SSc patients via IFN-γ production.

Primitive neuroectodermal tumor in an ovarian cystic teratoma: natural killer and neuroblastoma cell analysis.

In the present study, we report an extremely rare case of a 31-year-old woman with neuroblastoma arising in an ovarian cystic teratoma. We analyzed the expression of activating receptors on natural killer (NK) cells derived from the patient's peripheral blood and peritoneal fluid. In addition, we investigated the presence of specific ligands recognized by different NK cell receptors on tumor cells. We show that NK cells isolated from peritoneal fluid expressed certain triggering receptors including DNAM-1 (CD226) and CD16 with lower intensity as compared to peripheral blood NK cells. Remarkably, at variance with most cases of childhood neuroblastoma, the tumor cells from this patient expressed substantial amounts of HLA class-I molecules. These molecules are known to be protective against NK cell-mediated lysis. In addition, neuroblastoma cells expressed B7-H3 (CD276), another surface molecule that inhibits NK cell function. Finally, this tumor did not express the PVR (CD155) and nectin-2 (CD112) ligands for the DNAM-1 activating NK receptor, which plays a crucial role in NK/neuroblastoma interactions. Altogether, these findings indicate that the neuroblastoma cells of this patient express an NK-resistant surface phenotype, which is at least in part similar to that previously described in a fraction of childhood neuroblastoma.