Characterizing NK-like CD8+ T Cells in HIV-1 Chronically Infected Individuals Using Flow Cytometry

ABBREVIATIONS

MHC – Major Histocompatibility Complex

TCR – T Cell Receptor

iNKT – Natural Killer T

MAIT – Mucosal Associated Invariant T

NK – Natural Killer

KIR – Killer Immunoglobulin-like Receptor

IM – Innate Memory

VM – Virtual Memory

MPLA – Monophosphoryl Lipid A

PBMCs – Peripheral Blood Mononuclear Cells

ITIMs- Immunoreceptor Tyrosine-based Inhibitory Motifs

ITAMs – Immunoreceptor Tyrosine-based Activating Motifs

SLAM – Signalling Lymphocytic Activation Molecule

CMV –Cytomegalovirus

EBV – Epstein-Barr Virus

CML-CP – Chronic Phase of Chronic Myeloid Leukemia.

RRR – Pattern Recognition Receptors

PAMPs – Pathogen/damage Associated Molecular Patterns

pMHC – peptide-MHC Complex

IFN – Type I Interferon

SHP1 or 2 – Src Homology 2 domain containing Phosphatases

CRACC – CD2-like Receptor Activating Cytotoxic Cells

HIV – Human Immunodeficiency Virus

AIDS – Acquired Immuno Deficiency Syndrome

PD-1 – Programmed Death 1

KLRG1 – Killer Cell Lectin-like Receptor G1

Introduction 

The immune system has traditionally been subdivided into two arms, the innate and adaptive immune systems.The innate immune system is the first responder against foreign invaders, and acts rapidly in a non-specific manner to  clear infections as quickly as possible. This rapid response is triggered by signalling events that are initiated by Pattern Recognition Receptors (PRRs) that recognise Pathogen/damage Associated Molecular Patterns (PAMPs) or self-molecules. The innate immune system is  evolutionarily older than the adaptive immune systems, inherently present in most vertebrates and non-vertebrates, and is composed of physical and chemical barriers, phagocytic leukocytes, dendritic cells, natural killer (NK) cells, and plasma proteins¹. The adaptive immune system on the other hand takes a longer time to generate a response against an infection, but it is more potent and highly specific to the foreign invader than the innate immune response. A key feature of the adaptive immune system is that it has the capacity to develop immunological memory, which helps protect against recurrent infections. The adaptive immune system is evolutionarily a more recent development, found exclusively in vertebrates and consists of T cells and B cells¹. However, there has been a growing realization over the past two decades that the innate and adaptive immune systems are not as distinct as was initially believed. It is now known that there are several cell types that have functional characteristics of both systems, and that they essentially bridge these two branches. The existence of several subsets of unconventional T cells is an example of such cell types.

Conventional CD8+ T cells have constitutive cytotoxic activity and are responsible for killing infected/abnormal cells. They do this by recognising specific antigen peptides presented to them by cell-surface proteins of the Major Histocompatibility complex (MHC) I family, accompanied by co-stimulation and in the presence of appropriate cytokines. NK cells also have a crucial function in killing virus infected and tumour-transformed cells. However, unlike CD8+ T cells, NK cells utilize germ line encoded receptors that recognize missing self or altered self on the surface of a target cell in order to carry out their effector function. These NK cell receptors, which can be activating or inhibitory, are not restricted to NK cells and are also expressed on certain T cell subsets. These T cells are collectively known as unconventional T cells. They are not donor restricted, and can recognize antigens presented by non-polymorphic antigen-presenting molecules such as those encoded by genes within the MHC locus (HLA-E, HFE, H2-M3 and H2-Q9) or those encoded by genes outside the MHC locus (CD1a, CD1b, CD1c, CD1d and MR1)². Functionally, these unconventional T cells are poised to rapidly respond, and can be activated by both T cell receptor (TCR) ligation as well as danger signals and pro-inflammatory cytokines in a TCR independent fashion³. These populations of unconventional T cells include not only T cells, natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells but also the recently described human NK-like CD8 T cells⁴.

NK-like CD8+ T cells were first described in the early 2000s in studies conducted in both mice and humans, and were shown to be different to NKT cells which are CD1d restricted and have a semi-invariant TCR. These NK-like T cells co-expressed a TCR and natural killer (NK) cell receptors including CD56, killer cell immunoglobulin-like receptors (KIR), CD94, NKG2A, and NKG2C^5,6 (Figure 1). There is still a gap in the knowledge with respect to the specifics of the generation and differentiation processes associated with these cells but type I interferon (IFN), IL-4 and IL-15 have been shown to influence the same in mice⁴ <Sounds odd to me – influence the same in mice. What same?>. Studies in done in humans, on the role of these factors, are limited. There is more evidence in the literature regarding the phenotype of these cells in terms of the different receptors that these cells express, which shall be discussed further in the subsequent sections.

Figure 1: Diagrammatic comparison between a Conventional T cell and an NK-like T cell. Michel, J.J. et al. Front.Immunol.7, 530 (2016).

NK cell receptors

Prior to examiningthe phenotype of NK-like CD8+ T cells, it is important to understand the function of various NK cell receptors. This section takes a closer look at the various NK cell receptors commonly found on NK-like CD8+ T cells. The scope of the following discussion will, however, be limited to the receptors that were used as markers for the present study. Understanding NK cell receptors, their ligands, and general downstream signalling is of great importance given that NK cell responses are controlled by the balance of signalling through multiple activating and inhibitory cell surface receptors and would result in similar response when expressed on CD8+ T cells. NK cell receptors, unlike a TCR, are not highly antigen specific. They are germ line encoded, can be activating or inhibitory, or both, and are reported to be promiscuous with respect to ligand binding ⁷. They are grouped into families on the basis of similar recognition receptors as outlined below. Table 1 summarises the details of the various individual receptors within these families.

KIR Family Receptors

Members of the KIR family of receptors have evolved from the Ig-superfamily and consist of a trans-membrane domain, two or three Ig-like domains and a cytoplasmic tail ⁷.They can either by activating or inhibitory.  Inhibitory KIRs have been implicated in inhibiting cytokine release and target cell cytolysis in effector T cells⁸. Inhibitory KIRs signal through intracellular Immunoreceptor Tyrosine-based Inhibitory Motifs (ITIMs), which are located on the cytoplasmic tails of these receptors. On being stimulated, the receptor recruits Src homology 2 domain containing phosphatases (SHP1 or 2) followed by downstream signalling⁹. Activating KIRs signal through DAP-12 ⁷. While the exact sequence of signalling events are not well characterized, DAP-12 signalling are distinct, wherein the former results in cytotoxicity and the latter results in cytokine secretion and cytotoxicity¹⁰. KIRs specifically bind HLA-A, HLA-B and HLA-C and recognize polymorphisms in these molecules⁷. This binding of the KIR to the HLA molecule is also influenced by the peptide bound to the HLA molecule¹¹. Despite these similarities between KIR and TCR recognition of the peptide-MHC Complex (pMHC), the region of the ligands recognized by the two receptors, peptide specificity, the downstream signalling and physiological responses initiatedby both receptors are very different¹².

 

CD94-NKG2 Heterodimer Receptors

CD94-NKG2A/C/E heterodimers are a family of C-type lectin receptors. These receptors recognize non-classical MHC class I molecules (HLA-E in humans) on the surface of potential target cells¹³. HLA-E binds peptides derived from the leader sequence of MHC class I molecules on target cells and thus allows CD94-NKG2A and CD94-NKG2C to indirectly monitor the expression of MHC class I molecules on the cell¹³. CD94-NKG2A in humans is inhibitory and thought to be a key player in preventing inappropriate NK cell activation and inhibiting antigen-specific effector functions in T cells^8,14. The other receptors in this family are stimulatory, and whether or not they possess the ability to modulate T cell function is still not known¹⁵. In humans, expression of these receptors may be inversely related to KIR expression as NK cell clones that lack inhibitory KIR expression were shown to express an inhibitory CD94-NKG2 heterodimer¹⁶.

Signalling Lymphocytic Activation Molecule (SLAM) Receptors

Members of this family include 2B4 and CD2-like receptor activating cytotoxic cells (CRACC). They possess two Ig-like domains – one variable (V)-like domain and one constant 2 (C2)-like domain, a transmembrane domain and a cytoplasmic tail bearing multiple tyrosine-based motifs¹⁷. SLAM-related receptors are typically self-ligands with the exception of 2B4, which interacts with CD48 ¹⁷.The NK cell receptor CD244 (2B4) on being stimulated, recruits the adaptor proteins SAP or ERT, which contain Src homology 2 domains, by means of an immunoreceptor tyrosine-based switch motif present in the cytoplasmic tail¹⁸. This results in downstream signalling being initiated

Receptor	ligand(s)	Expression	Function
Killer immunoglobulin-like receptors (KIR) family
KIR2DL1 (p58.1)	HLA-Cw2, 4, 5, 6	NK, T cells	Inhibitory
KIR2DL2 (p58.2)	HLA-Cw1, 3, 7, 8	NK, T cells	Inhibitory
KIR3DL1 (p70)	HLA-Bw4	NK, T cells	Inhibitory
KIR3DL3 (p140)	HLA-A3, -A11	NK, T cells	Inhibitory
KIR2DS1 (p50.1)	HLA-Cw2,4,5,6	NK, T cells	Stimulatory
KIR2DS2 (p50.2)	HLA-Cw1,3,7,8	NK, T cells	Stimulatory
KIR2DS4 (p50.3)	Unknown	NK, T cells	Stimulatory
CD94-NKG2 heterodimers
CD94/NKG2A	HLA-E	NK, CTL	Inhibitory
CD94/NKG2C	HLA-E	NK, CTL	Stimulatory
CD94/NKG2E	HLA-E	NK, CTL	Stimulatory
SLAM receptor family
2B4 (CD244)	CD48	All NK cells ,a CD8+ T cell subset and monocytes	Inhibitory/Stimulatory
CRACC	Homophilic	CTL, activated B cells, NK cells and mature DCs	Inhibitory/Stimulatory

Table 1: Human NK cell receptors ligands and function¹⁹. Adapted from Wu, et al Front biosci10, 3132-3142 (2005).

 

Phenotypic characteristics of NK-like CD8+ T cells

NK-like CD8+ T cells have been described using many different names including CD56+ CD8+ NKT-like cells and KIR+ CD8+ T cells, Effector memory CD8+ T cells that express NK cell receptors have been referred to as innate like CD8+ T cells. This section attempts to look more closely at the various studies conducted on NK-like CD8+ T cells in both mice and humans in order to understand their phenotype.

CD8+ T cells producing IFN in response to innate signals in a TCR independent fashion were described in mice roughly at the same time as NK-like CD8+ T cells were being described in humans^4,20.  These cells possessed a memory phenotype (CD44+, CD62L-, CD122+) and were shown to provide protection against infections caused by Listeria monocytogenes²¹. This work was closely followed by the identification of a population of thymic CD8+ T cells called innate memory (IM) CD8+ T cells that responded to IL-12 and IL-18 by producing IFN^4,22. A similar population of cells were also described in mouse spleen and peripheral lymphoid organs, and were called virtual memory (VM) CD8+ T cells^4,23.  Both IM and VM CD8+ T cells possess a memory phenotype (CD44+, CD62L-) and their differentiation has been demonstrated to be dependent on Eomes and IL-15^22,23. IM and VM CD8+ T cells have been differentiated from each other on the basis of expression of the integrin CD49d by mouse thymic IM CD8+ T cells⁴. Wang et al. defined NK-like CD8+ T cells in mice asTCR+ CD3+ NK1.1+ CD49b+ NKG2D+ cells and described their ability to secreteIFN when stimulated with dendritic cells²⁴. Yet another study in mice defined NK-like CD8+ T cells purely on the basis of their memory phenotype (CD44+, CD62L-), and described the activation and expansion of these cells in response to co-administration of -GalCer and the TLR4 agonist monophosphoryl lipid A (MPLA)²⁵.

NK-like CD8+ T cells in humans were characterised by several studies as possessing a T_EMRA (CD45RA+, CCR7-, CD57+) phenotype^26. Evidence went on to show that these KIR+ CD8+ T_EMRAcells appeared to have fewer different V when compared to their negative counterparts²⁶. This skewing within the KIR+ CD8+ T_EMRA population is suggestive of such cells having undergone a phase of expansion after acquisition of the KIR, possibly as a result of prolonged antigenic pressure^4,26. These KIR+ CD8+ cells also had a weaker response to TCR stimulation than their KIR- counterparts in terms of expression of IFN, TNF and degranulation, which was evaluated by CD107a staining²⁶. This subset of cells however responded very well to innate cytokine stimulation with IL-12 and IL-18²⁷. Different groups have chosen to define NK-like CD8+ T cells differently in each of their respective studies. In a study looking at the expression of inhibitory NK-receptors in human melanoma–specific CD8+ T cells, melanoma antigen specific TCR+ CD8+ cells were studied and the percentage of NK-receptor positive (p58.1, p58.2, p70, p140, ILT2, NKRP1A, ZIN176, CD94 and CD94/NKG2A) T cells were assessed²⁸. These cells were shown to have anterminally differentiated phenotype (CD28-, CD57+)²⁸. Hassounehet al. chose to define NK-like CD8+ T cells as CD57+ CD56+ CD8+ T cells in his study looking at these cells in the context of Cytomegalovirus (CMV)²⁹. Yet another study on NK-like CD8+ T cells defined these cells as KIR+ and/or NKG2A+ CD8+ T cells and showed that they preferentially expressed Eomes, had a memory phenotype, and shared functional and phenotypic features with IM CD8+ T cells in mice³⁰. These cells also preferentially expressed CD57 and CD49d^4,30. CD49d is an α4-integrin, which is important in determining the localization of T cells to different territories based on the β chain to which it is matched⁴. In NK-like CD8+ T cells however, its use as a functional marker has been proposed making it a possible characteristic marker for these cells⁴. The aforementioned studies describe a variety of NK cell receptors that are expressed on T cells. It is however essential to realize that the NK cell repertoire that is expressed on NK-like CD8+ T cells express does not reflect the full complement of NK receptors expressed on NK cells³¹. While there is no clear cut definition of NK-like CD8+ T cells, it appears that they can be best defined as CD8+ T cells that express NK receptors, have impaired TCR signalling, display a memory phenotype, express Eomes and respond to innate cytokine stimulation (IL-12 and IL-18) by producing IFN  (see Figure 2).

Figure 1. Representative diagram of a human NK-like CD8+ T cell detailing the most commonly used markers and nuclear transcription factors that assist in defining this unique subpopulation of cells. These cells also respond to IL-12 and IL-18 innate-like stimulation and produce IFN. Adapted from Barbarin, A. et al.Front. Immunol.8, 316 (2017).

NK-like CD8+ T cells in Immunosenescence and Viral Infections

Immunosenescence refers to the faulty immune response seen during aging, that occurs because of general decline in immune function³⁸. Aging sees a steady decline in the number of naïve T cells emerging from the thymus, but this is accompanied by an increase in the number of memory T cells that undergo significant changes in phenotype and function especially CD8+ T cells^3,39. A hallmark characteristic of aging T cells is the loss of co-stimulatory receptors such as CD28 and CD27^4,40. This loss or absence of co-stimulatory receptors is believed to be the reason for deficiency or ineffective TCR signalling in aged T cells. An increase in the number of effector/senescent T cells expressing NK markers has also been reported to be associated with aging⁴¹. Evidence clearly shows that there is differential regulation of NK cell receptor expression between T cells and NK cells⁴². For example, expression levels of CD158b1 (KIR 2DL2) in NK cells and T cells have been reported as being controlled by two distinct transcriptional regulatory motifs on the upstream cis-acting promoter region of the gene⁴³. This suggests that expression of NK receptors on T cells is not a random event associated with aging, but that it is actually a physiologically programmed phenomenon. This acquisition of a diverse array of NK receptors by inefficiently functioning T cells, helps maintain immunologic diversity in old age. It also helps to compensate for any loss in the NK cell compartment, which might occur due to skewing of NK cell subsets with aging⁴⁴. It appears that the age-dependent appearance of functionally competent senescent NK-like CD8+ T cells represents an overall beneficial remodelling of the immune repertoire³⁸. It is however important to keep in mind that aging is a process that is unique to an individual and results in highly heterogeneous health and immune phenotypes being observed within a cohort being studied making extrapolatingand discussing results a challenge.

NK-like CD8+ T cells and Disease associations

An increase in percentage of frequency of NK-like CD8+ T cells has been described in individuals suffering from disease conditions such as Behcet’s Uveitis and Leishmaniasis<please check that I have corrected these names properly> where they produce more IFN- compared to healthy individuals, and in chronic obstructive pulmonary disease where they produce IFN and TNF^32-34. NK-like CD8+ T cells have also been described as potentially having an anti-cancer role. This can be inferred, indirectly, on the basis of studies conducted in patients in the chronic phase of chronic myeloid leukemia (CML-CP). These patients have been shown to have major defects in their NK-like CD8+ T cells when compared with healthy subjects or patients in complete remission. The defects manifest as a loss of IFN-γ expression in response to innate cytokine stimulation by IL-12 and IL-18,and loss of degranulation after stimulation via CD16³⁵. However, IFN-γ expression after TCR stimulation by the same NK-like CD8+ T cells during CP was conserved, which indicates that the functional defect affecting NK-like CD8+ T cells is innate rather than adaptive³⁵. The implications of this defect, with respect to understanding functionality of these cells, are yet to be investigated. The presence of these NK-like CD8+ T cells was also detected in solid tumours such as lymph nodes invaded by tumour cells in metastatic breast cancer, and amongst intra-tumoural lymphocytes in ovarian cancer⁴. These studies provide evidence that NK-like CD8+ T cells are present not only in tumours, but that they are also probably intimately integrated in the dynamics of anticancer responses.

NK-like CD8+ T cells in Epstein-Barr virus (EBV) associated tumour patients were shown to be quantitatively and functionally impaired³⁶. Women suffering from Human Papilloma virus (HPV) associated cervical neoplasia showed increased levels of CD28-, T_EM and CD16+ CD56+ CD8+ T cells amongst their PBMCs, which is probably a result of the chronic HPV infection³⁷.  From these studies it can be inferred that NK-like CD8+ T cells interact with other cells and their phenotype is greatly influenced by the type of chronic antigenic stimulation they experience.

Several reports state that one of the main drivers responsible for the accumulation of highly differentiated antigen specific CD8+ T cells that display characteristics of replicative senescence, is persistent viral infections such as CMV and EBV, which cause chronic immune activation^3,45,46.The studies pertaining to CMV however, are purely associative and not causal. Further, these studies involved a cohort of individuals that were belonged to an elderly population. Thus the causative role of CMV in inducing expansion of senescent NK-like CD8+ T cells remains to be proven. The current hypothesis being proposed is that NK-like CD8+ T cells accumulate with age in CMV seropositive individuals as opposed to CMV infection per se⁴⁷.  However, the expansion of CD57+ CD8+ T cells is a hallmark of latent CMV infection, where CD57+ T cells display a reduced capacity to proliferate accompanied with altered functional properties^48,49. Furthermore, NK-like CD8+ T cells in CMV seropositive individuals are also largely CD57+ thus implying that they are mostly senescent²⁹.

NK-like CD8+ T cells in HIV-1 infected individuals

 

The human immunodeficiency virus (HIV), which is responsible for causing Acquired Immuno Deficiency Syndrome (AIDS), is one of the world’s most serious health and development challenges. As of 2016, an estimated 36.7 million people worldwide were living with HIV/AIDS⁵⁰. Of these, 2.1 million were children (<15 years old). HIV-1 predominantly replicates in CD4+ T cells , both in the early and late stages of the disease. The early/primary stage of HIV infection sees titre levels of the virus reach peak levels; the viremia, however, stabilizes around 3 to 6 months later as a result of partially being controlled by the immune response⁵¹. CD8+ cytotoxic T cells are especially important in the clearance of HIV infected cells and help control the infection. However, CD8+ T cell responses in chronic HIV-1 infection are relatively impaired which results in inefficient clearance of the virus⁵².  The abnormalities/defects seen in HIV-1 infected CD8+ T cells are similar to those seen in CD8+ T cells during aging which is relatively unsurprising given that HIV infected individuals experience accelerated immunosenescence ⁵³. CD8+ T cells in HIV infected individuals express increased levels of the inhibitory receptor programmed death 1 (PD-1) and the killer cell lectin-like receptor G1 (KLRG1), which are markers for exhaustion and immunosenescence respectively^54,55.

A limited amount is known about NK-like CD8+ T cells in HIV-1 infected individuals. A few studies have described KIR+ CD8+ T cells in HIV-1 infected individuals as a population of cells that are largely non-responsive to TCR dependent stimulation^56-58. These studies have also demonstrated that KIR expression on CD8+ T cells is associated with a reduced ability to kill target cells. These cells have also been shown to predominantly be memory CD8+ T cells. Another study has gone on to describe a generalized suppression of TCR-mediated activity of KIR+ CD8+ T cells during HIV-1 infection via a ligand-independent blockade of TCR activation. The phenotype and function of these cells needs to be further assessed.

Project Aims

The main aim of this project was to define and phenotypically characterize NK-like CD8+ T cells in HIV-1 chronically infected individuals using flow cytometry on the basis of expression of NK cell receptors, memory phenotype, exhaustion markers, transcriptional profile and TCR usage. We also set out to understand their functional properties by investigating their response to innate cytokine stimulation. This project was aimed at providing insight into the relevance of these cells in the context of HIV infection and providing preliminary data towards investigating the possibility of these cells as an immunotherapeutic.

Bibiliography

1 Murphy, K. & Weaver, C. Janeway’s immunobiology.  (Garland Science, 2016).

2 Godfrey, D. I., Uldrich, A. P., McCluskey, J., Rossjohn, J.&Moody, D. B.The burgeoning family of unconventional T cells. Nature Immunology16, 1114, doi:10.1038/ni.3298 (2015).

3 Pereira, B. I. & Akbar, A. N. Convergence of Innate and Adaptive Immunity during Human Aging. Frontiers in immunology7, 445, doi:10.3389/fimmu.2016.00445 (2016).

4 Barbarin, A. et al. Phenotype of NK-Like CD8(+) T Cells with Innate Features in Humans and Their Relevance in Cancer Diseases. Frontiers in immunology8, doi:10.3389/fimmu.2017.00316 (2017).

5 Arlettaz, L., Degermann, S., De Rham, C., Roosnek, E. & Huard, B. Expression of inhibitory KIR is confined to CD8+ effector T cells and limits their proliferative capacity. European journal of immunology34, 3413-3422, doi:10.1002/eji.200324756 (2004).

6 Arlettaz, L. et al. Activating CD94:NKG2C and inhibitory CD94:NKG2A receptors are expressed by distinct subsets of committed CD8+ TCR alphabeta lymphocytes. European journal of immunology34, 3456-3464, doi:10.1002/eji.200425210 (2004).

7 Pegram, H. J., Andrews, D. M., Smyth, M. J., Darcy, P. K. & Kershaw, M. H. Activating and inhibitory receptors of natural killer cells. Immunol Cell Biol89, 216-224, doi:10.1038/icb.2010.78 (2011).

8 McMahon, C. W. & Raulet, D. H. Expression and function of NK cell receptors in CD8+ T cells. Current opinion in immunology13, 465-470 (2001).

9 Tomasello, E., Blery, M., Vely, F. & Vivier, E. Signaling pathways engaged by NK cell receptors: double concerto for activating receptors, inhibitory receptors and NK cells. Semin Immunol12, 139-147, doi:10.1006/smim.2000.0216 (2000).

10 Zompi, S. et al. NKG2D triggers cytotoxicity in mouse NK cells lacking DAP12 or Syk family kinases. Nat Immunol4, 565-572, doi:10.1038/ni930 (2003).

11 Parham, P. MHC class I molecules and KIRs in human history, health and survival. Nature reviews. Immunology5, 201-214, doi:10.1038/nri1570 (2005).

12 Rajagopalan, S. & Long, E. O. Antagonizing inhibition gets NK cells going. Proceedings of the National Academy of Sciences of the United States of America107, 10333-10334, doi:10.1073/pnas.1005636107 (2010).

13 Borrego, F., Ulbrecht, M., Weiss, E. H., Coligan, J. E. & Brooks, A. G. Recognition of human histocompatibility leukocyte antigen (HLA)-E complexed with HLA class I signal sequence-derived peptides by CD94/NKG2 confers protection from natural killer cell-mediated lysis. J Exp Med187, 813-818 (1998).

14 Braud, V. M. et al. HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature391, 795-799, doi:10.1038/35869 (1998).

15 Carretero, M. et al. The CD94 and NKG2-A C-type lectins covalently assemble to form a natural killer cell inhibitory receptor for HLA class I molecules. European journal of immunology27, 563-567, doi:10.1002/eji.1830270230 (1997).

16 Valiante, N. M. et al. Functionally and structurally distinct NK cell receptor repertoires in the peripheral blood of two human donors. Immunity7, 739-751 (1997).

17 Veillette, A. SLAM-family receptors: immune regulators with or without SAP-family adaptors. Cold Spring Harb Perspect Biol2, a002469, doi:10.1101/cshperspect.a002469 (2010).

18 Veillette, A. NK cell regulation by SLAM family receptors and SAP-related adapters. Immunological reviews214, 22-34, doi:10.1111/j.1600-065X.2006.00453.x (2006).

19 Wu, P., Wei, H., Zhang, C., Zhang, J. & Tian, Z. Regulation of NK cell activation by stimulatory and inhibitory receptors in tumor escape from innate immunity. Frontiers in bioscience : a journal and virtual library10, 3132-3142 (2005).

20 Berg, R. E., Cordes, C. J. & Forman, J. Contribution of CD8+ T cells to innate immunity: IFN-gamma secretion induced by IL-12 and IL-18. European journal of immunology32, 2807-2816, doi:10.1002/1521-4141(2002010)32:10<2807::AID-IMMU2807>3.0.CO;2-0 (2002).

21 Berg, R. E., Crossley, E., Murray, S. & Forman, J. Memory CD8+ T cells provide innate immune protection against Listeria monocytogenes in the absence of cognate antigen. J Exp Med198, 1583-1593, doi:10.1084/jem.20031051 (2003).

22 Atherly, L. O. et al. The Tec family tyrosine kinases Itk and Rlk regulate the development of conventional CD8+ T cells. Immunity25, 79-91, doi:10.1016/j.immuni.2006.05.012 (2006).

23 Lee, J. Y., Hamilton, S. E., Akue, A. D., Hogquist, K. A. & Jameson, S. C. Virtual memory CD8 T cells display unique functional properties. Proceedings of the National Academy of Sciences of the United States of America110, 13498-13503, doi:10.1073/pnas.1307572110 (2013).

24 Wang, C. et al. CD8(+)NKT-like cells regulate the immune response by killing antigen-bearing DCs. Sci Rep5, 14124, doi:10.1038/srep14124 (2015).

25 Coelho-Dos-Reis, J. G. et al. Co-administration of alpha-GalCer analog and TLR4 agonist induces robust CD8(+) T-cell responses to PyCS protein and WT-1 antigen and activates memory-like effector NKT cells. Clin Immunol168, 6-15, doi:10.1016/j.clim.2016.04.014 (2016).

26 Bjorkstrom, N. K. et al. CD8 T cells express randomly selected KIRs with distinct specificities compared with NK cells. Blood120, 3455-3465, doi:10.1182/blood-2012-03-416867 (2012).

27 Guia, S. et al. A role for interleukin-12/23 in the maturation of human natural killer and CD56+ T cells in vivo. Blood111, 5008-5016, doi:10.1182/blood-2007-11-122259 (2008).

28 Speiser, D. E. et al. In vivo expression of natural killer cell inhibitory receptors by human melanoma-specific cytolytic T lymphocytes. J Exp Med190, 775-782 (1999).

29 Hassouneh, F. et al. Effect of age and latent CMV infection on CD8+ CD56+ T cells (NKT-like) frequency and functionality. Mech Ageing Dev158, 38-45, doi:10.1016/j.mad.2015.12.003 (2016).

30 Jacomet, F. et al. Evidence for eomesodermin-expressing innate-like CD8(+) KIR/NKG2A(+) T cells in human adults and cord blood samples. European journal of immunology45, 1926-1933, doi:10.1002/eji.201545539 (2015).

31 Abedin, S., Michel, J. J., Lemster, B. & Vallejo, A. N. Diversity of NKR expression in aging T cells and in T cells of the aged: the new frontier into the exploration of protective immunity in the elderly. Exp Gerontol40, 537-548, doi:10.1016/j.exger.2005.04.012 (2005).

32 Yu, H. G. et al. The number of CD8+ T cells and NKT cells increases in the aqueous humor of patients with Behcet’s uveitis. Clinical and experimental immunology137, 437-443, doi:10.1111/j.1365-2249.2004.02536.x (2004).

33 Kumari, S. et al. Leishmania donovani skews the CD56(+) Natural Killer T cell response during human visceral leishmaniasis. Cytokine73, 53-60, doi:10.1016/j.cyto.2015.01.011 (2015).

34 Hodge, G., Hodge, S., Reynolds, P. N. & Holmes, M. Targeting peripheral blood pro-inflammatory CD28null T cells and natural killer T-like cells by inhibiting CD137 expression: possible relevance to treatment of bronchiolitis obliterans syndrome. J Heart Lung Transplant32, 1081-1089, doi:10.1016/j.healun.2013.07.017 (2013).

35 Jacomet, F. et al. The Hypothesis of the Human iNKT/Innate CD8(+) T-Cell Axis Applied to Cancer: Evidence for a Deficiency in Chronic Myeloid Leukemia. Frontiers in immunology7, 688, doi:10.3389/fimmu.2016.00688 (2016).

36 Xiao, W. et al. EBV-induced human CD8(+) NKT cells synergize CD4(+) NKT cells suppressing EBV-associated tumors upon induction of Th1 bias. Cellular and Molecular Immunology8, 368-368, doi:10.1038/cmi.2011.66 (2011).

37 Pita-Lopez, M. L., Ortiz-Lazareno, P. C., Navarro-Meza, M., Santoyo-Telles, F. & Peralta-Zaragoza, O. CD28-, CD45RA(null/dim) and natural killer-like CD8+ T cells are increased in peripheral blood of women with low-grade cervical lesions. Cancer Cell Int14, 97, doi:10.1186/s12935-014-0097-5 (2014).

38 Vallejo, A. N. Immune remodeling: lessons from repertoire alterations during chronological aging and in immune-mediated disease. Trends Mol Med13, 94-102, doi:10.1016/j.molmed.2007.01.005 (2007).

39 Linton, P. J. & Dorshkind, K. Age-related changes in lymphocyte development and function. Nat Immunol5, 133-139, doi:10.1038/ni1033 (2004).

40 Weng, N. P., Akbar, A. N. & Goronzy, J. CD28(-) T cells: their role in the age-associated decline of immune function. Trends Immunol30, 306-312, doi:10.1016/j.it.2009.03.013 (2009).

41 Tarazona, R. et al. Increased expression of NK cell markers on T lymphocytes in aging and chronic activation of the immune system reflects the accumulation of effector/senescent T cells. Mech Ageing Dev121, 77-88 (2000).

42 Vallejo, A. N. et al. Expansions of NK-like alphabetaT cells with chronologic aging: novel lymphocyte effectors that compensate for functional deficits of conventional NK cells and T cells. Ageing Res Rev10, 354-361, doi:10.1016/j.arr.2010.09.006 (2011).

43 Xu, J., Vallejo, A. N., Jiang, Y., Weyand, C. M. & Goronzy, J. J. Distinct transcriptional control mechanisms of killer immunoglobulin-like receptors in natural killer (NK) and in T cells. J Biol Chem280, 24277-24285, doi:10.1074/jbc.M500727200 (2005).

44 Le Garff-Tavernier, M. et al. Human NK cells display major phenotypic and functional changes over the life span. Aging Cell9, 527-535, doi:10.1111/j.1474-9726.2010.00584.x (2010).

45 Ouyang, Q. et al. Age-associated accumulation of CMV-specific CD8+ T cells expressing the inhibitory killer cell lectin-like receptor G1 (KLRG1). Exp Gerontol38, 911-920 (2003).

46 Ouyang, Q. et al. Dysfunctional CMV-specific CD8(+) T cells accumulate in the elderly. Exp Gerontol39, 607-613, doi:10.1016/j.exger.2003.11.016 (2004).

47 Pita-Lopez, M. L., Pera, A. & Solana, R. Adaptive Memory of Human NK-like CD8(+) T-Cells to Aging, and Viral and Tumor Antigens. Frontiers in immunology7, 616, doi:10.3389/fimmu.2016.00616 (2016).

48 Kared, H., Martelli, S., Ng, T. P., Pender, S. L. & Larbi, A. CD57 in human natural killer cells and T-lymphocytes. Cancer Immunol Immunother65, 441-452, doi:10.1007/s00262-016-1803-z (2016).

49 Pera, A. et al. CMV latent infection improves CD8+ T response to SEB due to expansion of polyfunctional CD57+ cells in young individuals. PloS one9, e88538, doi:10.1371/journal.pone.0088538 (2014).

50 UNAID. Fact sheet – Latest statistics on the status of the AIDS epidemic, <http://www.unaids.org/en/resources/fact-sheet> (2017).

51 Lieberman, J., Shankar, P., Manjunath, N. & Andersson, J. Dressed to kill? A review of why antiviral CD8 T lymphocytes fail to prevent progressive immunodeficiency in HIV-1 infection. Blood98, 1667-1677 (2001).

52 Alter, G. et al. Ligand-independent exhaustion of killer immunoglobulin-like receptor-positive CD8+ T cells in human immunodeficiency virus type 1 infection. Journal of virology82, 9668-9677, doi:10.1128/JVI.00341-08 (2008).

53 Deeks, S. G. HIV infection, inflammation, immunosenescence, and aging. Annual review of medicine62, 141-155, doi:10.1146/annurev-med-042909-093756 (2011).

54 Day, C. L. et al. PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature443, 350, doi:10.1038/nature05115 (2006).

55 Thimme, R. et al. Increased expression of the NK cell receptor KLRG1 by virus-specific CD8 T cells during persistent antigen stimulation. Journal of virology79, 12112-12116, doi:10.1128/JVI.79.18.12112-12116.2005 (2005).

56 Sirianni, M. C. et al. Distribution of the natural killer-related receptor for HLA-C during highly active antiretroviral therapy for human immunodeficiency virus infection. Human immunology62, 1328-1334 (2001).

57 Galiani, M. D. et al. Expression of killer inhibitory receptors on cytotoxic cells from HIV-1-infected individuals. Clinical and experimental immunology115, 472-476 (1999).

58 Anfossi, N. et al. Coordinated expression of Ig-like inhibitory MHC class I receptors and acquisition of cytotoxic function in human CD8+ T cells. J Immunol173, 7223-7229 (2004).