The Expanding Role of Natural Killer Cells in Type 1 ... · The Expanding Role of Natural Killer Cells in Type 1 Diabetes and Immunotherapy ... direct T-cell responses and inhibit

PATHOGENESIS OF TYPE 1 DIABETES (A PUGLIESE, SECTION EDITOR)

The Expanding Role of Natural Killer Cells in Type 1Diabetes and Immunotherapy

Chris Fraker1 & Allison L. Bayer1,2

# Springer Science+Business Media New York 2016

Abstract Treatments for autoimmune diseases including type1 diabetes (T1D) are aimed at resetting the immune system,especially its adaptive arm. The innate immune system is oftenignored in the design of novel immune-based therapies. Thereis increasing evidence for multiple natural killer (NK) subpop-ulations, but their role is poorly understood in autoimmunityand likely is contributing to the controversial role reported forNKs. In this review, we will summarize NK subsets and theirroles in tolerance, autoimmune diabetes, and immunotherapy.

Keywords Natural killer cells . Diabetes . Tolerance

AbbreviationsNK Natural killer cellTeff T effector cellTregs T regulatory cellsT1D Type 1 diabetes

Introduction

Natural killer cells (NKs) belong to the innate arm of theimmune system and have critical roles in immune defense,

surveillance, and homeostasis. They act as a first responseagainst infections and tumor development through their cyto-toxic activity. However, NKs can also regulate many facets ofthe adaptive immune response by promoting inflammationthrough secretion of cytokines, such as IFNγ and TNFα, todirect T-cell responses and inhibit T regulatory cells (Tregs).Moreover, NKs regulate inflammation by killing antigen-presenting cells and activated T cells and producing anti-inflammatory cytokines, such as IL-10 [1]. NK recognitionand activation are mediated by the triggering of multiple in-hibitory and activating receptors, which influence whether asurveilled cell is protected or killed by an NK cell. Binding ofproinflammatory cytokine receptors also directs NK activityand function, which influences downstream immune re-sponses. Therefore, NKs provide a critical link between theinnate and adaptive immune system with the local environ-ment likely fundamental in determining functional outcomesof NK activity.

Human NKs in peripheral blood are characterized based onCD56 and CD16 expression. Most are defined asCD56dimCD16pos and have potent cytotoxic activity, killingtarget cells identified as Bnon-self^ or producing cytokines tostimulate further response; these are commonly called NKef fec to r (NKe f f ) c e l l s [2 ] . On the o the r hand ,CD56brightCD16neg NKs are predominately found in peripher-al tissues, albeit are also detected in blood; they respond tosoluble factors by making copious amounts of diverse immu-noregulatory cytokines, including IFNγ, TNFα, IL-10, andGM-CSF; these are termed regulatory NKs (NKreg) [3, 4].Conversely, mouse NKs lack CD56 expression, which hasmade it difficult to identify functional counterparts to humanNKs. The NKp46 (CD335) activation receptor is a commonsurface marker found on both human and mouse NKs [5–7].Together with CD122 (IL-2Rβ chain), these markers are usedto identify NKs among CD45posCD3neg cells. Many studies

This article is part of the Topical Collection on Pathogenesis of Type 1Diabetes

* Allison L. [email protected]

1 Diabetes Research Institute, University of Miami Miller School ofMedicine, Miami, FL 33136, USA

2 Department of Microbiology and Immunology, University of MiamiMiller School of Medicine, Miami, FL 33136, USA

Curr Diab Rep (2016) 16:109 DOI 10.1007/s11892-016-0806-7

http://crossmark.crossref.org/dialog/?doi=10.1007/s11892-016-0806-7&domain=pdf

have examinedmouse NKs based on the expression of CD11band CD27, but these markers focus more on immature versusmature status during mouse NK development and less on theirfunctional capacity, especially as it relates to disease status.Furthermore, recent studies have identified distinct tissue-resident (tr)NKs that are CD49aposDX5neg and have differentcytokine production and developmental requirements [8, 9••,10••]. How these different NK subsets participate in shapingdownstream immune responses in their microenvironmentsand systemic immune responses is still largely unknown inmany disease states.

Type 1 diabetes (T1D) is an organ-specific autoimmunedisease caused by defective immunoregulation and selectivedestruction of insulin-producing pancreatic beta (β) cells.Disease development and progression are primarily causedby T effector (Teff) cells, but NKs have been detected in thepancreas of patients with T1D [11] and in experimental mousemodels of T1D [12–15], suggesting a participating role ofpancreas-infiltrating NKs. However, there are conflicting re-ports about NK involvement in autoimmune diabetes modelsdescribing both protective and destructive functions. Moststudies only examined bulk NK populations, which could ex-plain the controversy surrounding the role of NK cells ob-served in islet autoimmunity. The clarification of functionalNK subsets and their inflammatory and regulatory phenotypescould elucidate their part in T1D and other autoimmune dis-eases. Importantly, until only recently, NKs have beendisregarded in large part as a contributing factor in autoim-mune pathologies, including T1D. Instead, the majority ofresearch has focused on downstream adaptive immune path-ways and their primary players, such as CD8pos cytotoxic Tcells. With the recent work in NKs uncovering that they, liketheir adaptive counterparts, havemultiple subpopulations withgreat functional diversity, research into these upstream innatecells is intensifying.

Natural Killer Cell Development

The circulating conventional (c)NKs were thought to be theonly innate effector population but many new subsets of in-nate effector cells, termed innate lymphoid cells (ILCs), havebeen discovered (for review [16–18]). The cNKs belong to thegroup 1 ILC because they secrete IFNγ and are cytotoxic. ThecNKs primarily develop in the bone marrow and arise aseither NK precursor (NKP), immature, or mature cells thatdisperse throughout the body. The cNKs are derivedfrom the common lymphoid progenitor (CLP) cell, definedas LinnegSca-1lowCD117lowCD127posCD135pos. The firstdevelopmental stage is the NKP cell(s) defined asLinnegCD27posCD244posCD117lowCD127posCD135negC-D122pos in mice. This is followed by immature NKs withfurther maturation commonly characterized by CD27 and

CD11b expression [19]. The least mature NK population isCD27lowCD11blow, which does not express the late matura-tion marker DX5 (CD49b), or CD43. In humans, this popula-tion represents a tissue-resident cell that is critical in the main-tenance of pregnancy, referred to as decidual or tolerant NKs;however, a percentage of these cells express CD27 as well[20]. This immature CD27lowCD11blow expression is follow-ed by CD27highCD11blow, CD27highCD11bhigh, andCD27lowCD11bhigh, at which stage these most mature cellsexpress CD122, NKp46, DX5, KLGR1, and CD43 at highlevels. The development of cNKs requires the transcriptionfactor NFIL3. Functionally, immature CD27posCD11blow

NKs are potent cytokine producers but are less efficient killersthan the intermediate CD27posCD11bhigh subset and are abun-dant in the lymph nodes and bonemarrow, whereas the matureCD27negCD11bhigh NKs are enriched in the blood, spleen,liver, kidneys, and lungs. Mature NKs can efficientlylyse targets and produce proinflammatory cytokines afterstimulation with target cells or incubation with IL-12and IL-18. Collectively, these observations suggest thatmost mature NKs are a homogeneous population oflymphocytes, but recent evidence suggests the opposite,a heterogeneity among NKs in both mouse and man. Asan example, both mouse and human NKs have subpop-ulations (Fig. 1) that have varied production of proin-flammatory cytokines and cytotoxic activity based onthe expression of CD27 and CD11b [19–23].

HumanNKs are similarly derived from the CLP cell but arecategorized with differentiation points designated as develop-ment stages 1–5. The recently identified human NKP, definedas LinnegCD34posCD38posCD123negCD45RAposCD7pos

CD10posCD127neg, selectively gives rise to NKs in vitro andin vivo, but not to non-NK ILCs, T cells, B cells, or myeloidcells [24]. Cells in the least mature stages are CD56neg andCD94neg and become responsive to IL-15 through expressionof CD122. Stage 4 and 5 cells acquire CD56 expression andare now considered to have differentiated into the NK cell.Stage 4 cells are CD56brightCD16neg, produce high levels ofcytokines and low cytotoxic function, and are considered im-mature. Stage 5 cells are CD56dimCD16pos and potent targetcell-induced cytokine producers and cytotoxicity and are con-sidered mature. Recently, NKs expressing CD57 have beenidentified as a functionally distinct subset in the final stages ofperipheral NK maturation characterized by progression fromCD56bright to CD56dimCD57neg to CD56dimCD57pos cells[25–27]. CD57 expression in NKs represents a transition toincreased cytotoxic function and sensitivity to signalingthrough CD16 with decreased responsiveness to cytokines.Expression of CD57 increases with age and is also associatedwith chronic viral infections. Furthermore, the presence ofCD57pos mature NKs is consistently associated with betteroutcomes in cancer [28–33]. Autoimmune disease is associat-ed with reduced frequencies or absolute numbers of

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circulating CD57pos mature NKs [34–39], while the targetedtissues have local expansion of CD57negCD56dimCD16pos

NKs. However, it is currently unknown how proinflammatoryor pathogenic lymphocytes can affect these cells or the micro-environment to influence downstream immune responses.

Tissue-Resident Natural Killer Cells

NKs have long been thought of as a homogenous population oflymphocytes different from B- and T-cell compartments withtheir highly diverse antigen-receptor specificities and function-al responses. However, there is now clear evidence that NKrecognition and activation is highly diverse [8, 9••, 10••,40–44]. Several distinct NK populations have been identifiedin various tissues in humans and mice. Location of these tissue-resident NK (trNK) cells reveals unique phenotypic featureswith apparently distinctive development requirements. In the

mouse, a unique thymic NK subset exists which is phenotypi-cally different from cNK cell but shares some features withimmature mouse cNKs. Thymic NKs are CD127pos (IL-7Ra)but also express CD69highLy49lowCD11blow and are less cyto-toxic and produce more IFNγ, TNFα, and GM-CSF than theirsplenic cNK counterparts. Importantly, unlike the NFIL3- andIL-15-dependent cNK cells, thymic NK development requiresthe transcription factors GATA-3 and IL-7. Further, they do notdevelop from committed T-cell precursors. Interestingly, thy-mic NKs can traffic to the lymph nodes but not other tissues,and several reports have also shown that murine CD127pos

NKs may be similar to CD56brightCD16neg human NKs in thatthey express CD127, produce large amounts of cytokines, andonly acquire functional cytotoxicity after prolonged activation[45–47].

NKs are also found in peripheral tissues including the liver,lungs, skin, pancreas, and uterus in bothmice and humans [48,49]. However, only recently have trNKs been described to be

Fig. 1 Phenotype of NK subsets in human and mouse. Currentlydescribed NK subpopulations in human and mouse designated by CDsmost utilized in published literature: CD57 observed in all subpopulations

in our work (unpublished) with varying signal intensity (MFI). Function,in terms of cytokine secretion and cytotoxicity, represented by colorgradations on the right

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phenotypically distinct from circulating conventional splenicNKs and express many markers that resemble immature NKs(CD11blowDX5neg NKs) [8]. Detailed analysis shows that livertrNKs mutually express the DX5 (CD49b) maturation markerand CD49a (α1 integrin). Liver trNKs are CD49aposDX5neg,wh i l e sp len ic cNKs a re CD49an e gDX5po s . TheCD49aposDX5neg trNKs are also detected in the skin, kidney,uterus, and pancreas and are critically dependent on the tran-scription factor T-bet, while the development of cNKs is lessaffected by the T-bet deficiencies [9••, 50, 51]. HumanCD56brightCD16neg trNKs can also be identified through theexpression of CD69, CD103, and CD49a; this allows for thediscrimination of trNKs from the circulating CD56dimCD16pos

cNKs that are temporarily recruited to the tissues during im-mune responses, as almost all peripheral blood NKs lack thesemarkers [9••, 48, 49, 52, 53]. The developmental requirementsand the role of these human NK subsets are currently notknown, especially in regard to their dependence on particulartranscription factors such T-bet. Much of what is known abouthuman trNKs comes from studying these cells during patho-logical states; therefore, future studies should be aimed at un-derstanding NK subsets in healthy, peripheral tissues.Additionally, phenotypic characterization and functional stud-ies coupled with their anatomical location within the microen-vironment in both mouse and human would help reveal impor-tant cell-to-cell interactions and improve our understanding oftheir roles in local immune response and autoimmune diseases,which is largely unknown.

A recent study examining the roles of trNK and cNKs inthe kidney with ischemia and reperfusion injury (IRI) modeldemonstrated that trNKs are potent mediators of IRI [10••].This study uncovered that Asialo-GM1 expression was vari-able and reduced on trNKs and these cells were largely sparedfrom anti-Asialo-GM1 antibody depletion, while administra-tion of the depleting NK1.1 antibody (PK126) was effective atdepleting both cNK and trNKs leading to protection in thismodel. The lower Asialo-GM1 expression on trNKs was sim-ilar across tissues and mouse strains, and trNK frequencyamong total bulk NKs was found to be comparable acrossall mouse strains examined (B6, BALB/b, 129, and NOD)with the NOD showing slightly higher frequency in someorgans notably the pancreas [10••]. These findings have im-portant implications for future studies aimed at understandingthe role of NK subsets in autoimmunity asmany studies utilizethese two different antibodies for NK depletion strategies.Careful examination of not only the blood and secondarylymphoid tissue but also the targeted tissue needs to be exam-ined to determine trNK and cNK depletion. Moreover, studieswill need to avoid the use of the DX5 marker to identify bulkNKs, since trNKs lack this expression and this could lead to anoverestimation of NK depletion. The nonobvious complexityof NK subset depletion using these two strategies could becontributing to the controversial role observed for NKs in

autoimmune models. Future experimental design will needto incorporate delineation of these NK subsets to resolve theprotective and inflammatory role of NKs in autoimmune pa-thologies, including T1D.

NK Cells During Pregnancy: Tolerance Inductionand Reduced Cytotoxicity

In human beings, the fetus is protected from themother’s innateimmune response by a unique subset of CD56bright NKs thatpromote a tolerogenic environment in the decidua that aids inthe rich vasculature development of the uterus. Important inestablishing this microenvironment is the reduction ofCD56dimCD16pos NK cytotoxicity. This is accomplished pri-marily through the hyperexpression of the nonclassical majorhistocompatibility complex (MHC) class 1 membrane-boundHLA-G and elevated levels of its soluble isoforms. Themembrane-bound HLA-G isoform binds CD56dimCD16pos

NKs and reduces their cytotoxic capacity. Additionally, HLA-G serves to stabilize membrane HLA-E, another potent inhib-itor of NK cytotoxicity. Further, soluble HLA-G plays a role insignaling and trafficking of suppressive NKs that are abundantin the placental decidua during the first two trimesters of preg-nancy, known as decidual NKs. This NK subset, described inprior literature as CD56brightCD27negCD11bneg, produces thetolerogenic molecules (IL-10, TGFβ, VEGF). Interestingly,membrane-bound HLA-G has restricted tissue expression inthe adult body in trophoblast cells of the placenta, but it hassubsequently been found in erythroblasts and thymic epithelial,corneal, mesenchymal stem cells, and most intriguingly, pan-creatic islet β cells [54–59].

Some viruses (and other somatic invaders, like specificcancers) have adopted the aforementioned mechanisms toavoid detection by NKs and other components of the innateimmune system. Viruses will utilize, much like cancer andtrophoblast cells, the host’s own signaling pathways to disableinnate response [60–65, 66•, 67]. Viruses have been suggestedas a causative agent in many autoimmune pathologies includ-ing multiple sclerosis, T1D, Sjogren’s syndrome, rheumatoidarthritis, and systemic lupus erythematosus. Chronic NK de-fects, both in enumeration and function, have been observedin our own unpublished data and in many other studies exam-ining autoimmune patients and controls. Viral manipulation ofthis innate population would lead to compensatory responsesin an attempt to clear the pathogen, and this could explain theamplified adaptive response and unchecked inflammation as-sociated with T1D and many autoimmune pathologies.Additionally, in women between the ages of menarche andmenopause, the innate system and NKs are Bsuppressed^ eachmonth in preparation for fertilization and implantation. Thismight help explaining the huge disparity in the ratio of femaleto male autoimmune incidence.


Human NK Cells and Type 1 Diabetes

The exact role ofNKs in T1Dpathogenesis is still undefined.Wehypothesize that this may be due to the fact that prior literatureinvestigating NKs in T1D examines primarily bulk NKs anddoes not distinguish between NK subsets and their diverse func-tional differences. Many generally agree, however, that NK dys-function may contribute to T1D pathogenesis. Indeed, patientswith T1D and NOD mice are known to have aberrant NKG2Dsignaling, a common activating NK receptor, regardless of dis-ease duration, which suggests an involvement in the diseasepathogenesis [68]. Moreover, CD56dimCD16pos NKs are re-duced at T1D onset [69–71] with diminished responses to IL-2and IL-15 and lipopolysaccharide (LPS) and diminished cyto-toxicity and defective IFNγ secretion [68]. This reduction linksinto the long debated hypothesis that T1D and other autoim-mune pathologies may have a viral etiology, as they can directlyimpact NK function and enumeration.

The Evidence for Viral Etiology Is Growing

While a virus role has been debated for decades, evidence isbuilding with improved detection strategies and strong collab-orative efforts. Several findings support the association ofsome viral entities with T1D [11, 66•, 72]. Extensive researchhas provided epidemiological evidence for viral associationwith enteroviruses, rotavirus, and other types, but not all stud-ies have shown a link. Several methodological issues (samplesize, sampling frequency, assay sensitivity, biology of viralinfections) could explain some of the discordant results.However, other studies have clearly linked genetic factors thatcontrol T1D risk with viral infection. Experimental studieshave shown that enteroviruses infect β cells and cause dys-function [73]. Acute, lytic infections can induce β-cell death,and combined these effects could lead to the triggering ofchronic islet autoimmunity in genetically predisposed individ-uals. Pancreas specimens from patients recently diagnosedwith T1D from a UK cohort revealed the presence of viralantigens and inflammation in islets containing insulin-positive cells, a finding that has been noted at significantlyhigher frequency in patients with T1D compared to nondia-betic subjects [73]. Recent clinical studies also identified an-tibody responses to selected enterovirus strains as being asso-ciated with T1D [74]. Improved detection methodologies arehelping to unravel the association of viruses with T1D andother autoimmune conditions [75].

Critically, the mapping of a T1D susceptibility locus to theinterferon induced with helicase C domain 1 (IFIH1) geneprovides a genetic basis for a role of viruses in T1D[76–78]. IFIH1 encodes for a helicase (melanomadifferentiation-associated gene 5,MDA5), involved in the rec-ognition of double-stranded RNA, following the intracellularreplication of enteroviruses. This recognition results in

antiviral responses that are more intense in those carryingT1D-predisposing alleles at the IFIH1 locus. In T1D studiesin humans and animal models, autoimmune responses to βcells associated with elevated MDA5 expression.Conversely, lower MDA5 expression confers protection inmodels of disease and in at-risk human subjects. In humangenome-wide association studies (GWAS), specific polymor-phisms of this gene have demonstrated almost complete pro-tection against the development of T1D in those with high-risk association with other genetic markers. More sustainedinflammation would predictably amplify the potential to ex-pose self-antigens and trigger autoreactive responses and neg-atively impact β-cell function, including β-cell apoptosis in-duction [79]. Those with IFIH1 variants that protect from T1Dexhibit less intense inflammation following viral infection[77]. To our knowledge, to date, there are no reports that haveexamined MDA5 in relation to NK function.

Another paper presented strong evidence of improper viralclearance in T1D development; the study examined donorpancreata from six patients with T1D and 26 controls for signsof viral infection and lymphocyte infiltration [11]. In three ofthe six patients with T1D, Coxsackie B4 infection was detect-ed by staining of viral capsid protein (VP1) and then DNAextraction and sequencing of infected regions. In the samepatients, the islet infiltrates were comprised primarily ofNKs. In the other three patients with T1D, no virus was ob-served; infiltrates were NK-free and dominated mainly byCD8pos T cells more typically associated with T1D islet infil-trates. It is unlikely that at the time of death, all three patientshad ongoing Coxsackie B4 infection or that the cause of deathwas fulminant Coxsackie infection. Rather, this data offersadditional evidence for inefficient enterovirus clearancehighlighted by subclinical/latent infection and NK presence.As subset characterization was not performed, it is unclearwhether those NKs had cytotoxic function or were regulatory.Viruses, much like tumors, can recruit regulatory NKs that arefunctionally immunosuppressive like Tregs. The observed NKscould quite possibly be from this unique population; theycould also be dysfunctional NK effectors. Taken together,these studies lead to one hypothetical etiology for T1D: NKinability to clear viral infections leads to chronic infection andinnate immune suppression. In genetically susceptible indi-viduals with an amplified viral response, this phenotype leadsto an aberrant adaptive response and elevated inflammationcharacteristic of T1D.

NK Cells and Experimental Autoimmune Diabetes

The role of NKs in autoimmunity has been controversial withstudies showing either disease-promoting or disease-controlling roles. NKs are thought to participate in initiationof autoimmunity because they increase in target organs and


produce proinflammatory cytokines [11–14, 80–83].However, several reports have also shown protective effectsin experimental models of arthritis, colitis, and multiple scle-rosis [84–86]. In the context of viral etiology and manipula-tion of NKs through pregnancy tolerancemechanisms, it is notsurprising to see immunosuppressive NKs in the target tissue.NKs are a heterogeneous population and the downstream ef-fects in the local and systemic environment are not under-stood; again, this is confounded by the lack of detailed char-acterization of NK subsets, including cNKs and trNKs, andtheir functional capacity in the microenvironments in experi-mental models.

The first reports demonstrating a role for NKs in autoim-mune diabetes were in diabetic and diabetes-prone rats inwhich NKs were found to be more cytotoxic than NKs indiabetes-resistant rats [87–89]. In mice, antibody-dependentdepletion of NKs could prevent diabetes development inlow-dose streptozotocin (STZ)- or cyclophosphamide (CyP)-induced diabetes [90, 91]. Furthermore, when destructive pan-creatic infiltrates were compared to innocuous infiltrates, themain difference observed was increased NK proportions andincreased NK-specific transcripts in destructive lesions; NKdepletion significantly inhibited diabetes development [15].Alba et al. demonstrated NK pancreatic accumulation in anaccelerated diabetes model induced by overexpression ofIFNβ in pancreatic β cells in NOD, and NK depletioncompletely abolished this accelerated diabetes [92]. Feuereret al. showed that infiltrating pancreatic NKs play a criticalrole in the development of T1D in NOD mice and these NKspotently produced IFNγ and exhibited some cytotoxic activity[81]. In contrast, NK activation with complete Freund’s adju-vant protects against diabetes through the secretion of IFNγ inNODmice [93]. Another study in NODmice identified a newsubset of NKs with regulatory properties by inducing lysis ofactivated CD8 cytotoxic T lymphocytes (CTL) [94]. TheseNKs were generated from cNKs by IL-18 stimulation andexpressed c-Kit and programmed death (PD)-ligand 1; adop-tive transfer of these IL-18-st imulated NKs intostreptozotocin-treated C57BL/6 mice led to a delayed diabetesdevelopment and partial disease prevention, confirming a pro-tective role. Commonly, these studies focused on bulk NKs byexamining CD49b (DX5) expression, which is found on mostmature cNKs but was recently discovered to be absent ontrNKs. Moreover, most NK depletion strategies included ei-ther the administration of NK1.1 (PK126) or Asialo-GM1antibodies, which has recently been shown to lead to dispro-portionate depletion of NK subsets [10••]. Although the afore-mentioned studies noted effective NK depletion, an underes-timation of NK reduction likely occurred because of the ex-clusive CD49b examination. The use of NKp46 marker foridentification of bulk NKs (found on both cNKs and trNKs[10••]) together with CD49b and CD49a expression would bea more accurate measure of NK depletion, including cNK and

trNK subsets, and assist in clarifying the protective or destruc-tive role these NK subsets play in diabetes development.

A more detailed study of NKs (CD335posTCRβneg orCD3neg) in spontaneous diabetes was conducted in NODmice[12, 95]. Bulk NKs accumulated in the pancreas and displayedan active phenotype and proliferated more than the other tis-sues examined. Pancreatic NKs expressed higher levels ofCD69 and KLRG1 and lower levels of CD49b, CD43, andNKp46, and maturation status based on CD27 and CD11bexpression showed some differences compared to spleniccNKs. Ex vivo pancreatic NKs expressed less surfaceCD107a and produced less IFNγ than splenic NKs or pancre-atic NKs from C57BL/6 mice. Bielke et al. demonstrated thatNKs were not involved in diabetes development through ad-ministration of NK1.1 depleting antibody into several cohortsof NK1.1 NOD mice beginning at 2 weeks of age for eightconsecutive weeks. Diabetes onset was not significantly al-tered compared to control-treated mice. Interestingly, it wasnoted that recovery of peripheral NKs occurred 2 weeks afterthe end of treatment, but the phenotype or functional capacityof the recovering NKs in the blood or tissues was not exam-ined. It would be informative to know what NK subsetsreappeared after the depletion and whether differences in func-tion were observed, as it is possible that a protective NK ratherthan a destructive NK population now dominated the localmicroenvironment. Although the authors did examine the T-cell compartment at the very end of the antibody treatmentwith no major difference noted, it is not known whether T-cell populations were altered after the NKs recovered orwhether they were potentially modulated by regulatory NKs.Given that expression of Asialo-GM1 expression is varied andlower on trNKs in NOD mice, including in the pancreas[10••], depletion with Asialo-GM1 antibody could have pro-vided additional insights on the role of trNKs versus cNKs inautoimmune diabetes.

NKp46 (CD335 or NCR1) is a member of the natural cy-totoxic receptor (NCR) family and is considered one of themost NK-specific markers. NCR1 knockout mice on theC57BL/6 background were generated through insertion ofthe reporter gene GFP into the NCR1 locus. NCR1gfp/gfp B6mice are viable and contain NKs (GFP+) that lack NCR1-dependent functions including graft-versus-host disease [96],preventing tumor metastasis [97], and defense against patho-gens [98]. More recently, the first mechanistic evidence forNKs and diabetes was the discovery that β cells from NODmice and healthy humans express an unknown ligand for theNKp46 receptor [14]. The expression of the NKp46 ligand isstable in functional β cells and therefore β cells are constantlyat risk of being attacked by NKs through NKp46-stimulatedactivation. In vitro, incubation of NKs with β cells leads toNK degranulation and β cell killing. The NKp46 receptor wasalso found critical in diabetes development in vivo in low-dose STZ-diabetes in B6 mice. In this study, NOD mice were


treated with an NKp46-fusion protein that binds the ligand;this treatment results in the production of NKp46-specific an-tibodies, NKp46 downregulation, reduced NK killing activity,and in turn inhibition of diabetes development [14]. Undernormal conditions, NKs do not encounter β cells, but NKsare found in the immune infiltrates as disease progresses inNOD mice and can contribute to β cell loss. Further studiesare warranted using these mice to study NK subsets and theirfunctional roles and justify the need to backcross onto theNOD background to further delineate the role of NKs in au-toimmune diabetes.

Interplay of Innate and Adaptive Immunity: NK, T,and T Regulatory Cells and IL-2

Autoimmunity is in part due to an imbalance between Teff andTregs. Immunotherapies in this context are aimed at re-establishing this balance to promote long-term protection fromdestructive autoimmune responses. T1D is caused by the au-toimmune destruction of insulin-producingβ cells by infiltrat-ing immune cells. Both innate and adaptive immune cells havebeen shown to participate in disease pathogenesis with T andB cells, but also NKs, dendritic cells, and macrophages.Clinical trials in T1D as well as other immune-mediated dis-eases are testing low-dose IL-2 (LD IL-2) to selectively stim-ulate Tregs and their regulatory function. Other strategies in-volve adoptive transfer of autologous Tregs with severalgroups taking the approach of ex vivo Treg expansion and haveestablished expansion protocols for clinical trials. Clinical tri-als with autologous, expanded Tregs are ongoing in T1D. Dataemerging from T1D trials with these autologous Tregs and intransplant patients with allogeneic Tregs demonstrate the limi-tations of protocols that rely on expanded Tregs infusion with-out any recipient manipulation. Data from our laboratory hasshown the importance of host environment as a key determi-nant of Treg engraftment and therapeutic efficacy. We haveaddressed key questions about the environment and modeledstrategies that facilitate long-term engraftment and function ofTreg therapeutic infusions ([99–102] or review [103]).However, the role of NKs in this context is not known andneeds to be understood in order to improve therapeutic strat-egies aimed at resetting the balance between Teff and Tregs.

Tregs play a critical role in NK homeostasis, activation, andfunction, predominantly by controlling the availability of IL-2in the microenvironment. Data from the Mathis group showthat in the absence of Treg cells, NKs produce abundant IFNγand contribute to the diabetic lesion in NOD mice. NKs werefound to be initiators of autoimmune responses by stimulatingCD4 Tcells [81]. Moreover, IL-2 was the critical link betweenTreg and NKs in the pancreatic microenvironment [104•]: bulkNKs (CD3negCD335pos) were examined and higher percent-ages of IFNγ-producing NKs were found. A more detailed

examination of NKs following targeted Treg depletion in B6mice found that a CD127pos (IL-7Rαpos) NK subset increasesfollowing Treg depletion and contains immature NKs that ex-press c-Kit and CD25, produce IFNγ, and exhibit low cyto-toxic activity [105•]. Moreover, CD127pos NKs were in-creased in tumor-bearing and chronically infected mice, indi-cating that this NK population was not a unique feature ofautoimmunity but rather is present in chronic inflammatoryconditions. The expansion of CD127pos NKs was dependenton activated CD4 T cells and increased availability of IL-2,due to the loss of Tregs.

The IL-2 pathway is genetically impaired in NOD mice,which is also accompanied by reduced IL-2 levels [106–108].Bluestone and colleagues have shown the important role ofIL-2 in the NOD pancreatic microenvironment for Treg surviv-al and function [109, 110]. At diabetes onset, there are Tregapoptosis, an altered Teff/Treg ratio, and increased IFNγ pro-duction exacerbating autoimmunity in pancreatic microenvi-ronment which was corrected with LD IL-2. Collectively,these studies suggest that prior to disease onset, IL-2 is utilizedprimarily by Tregs, allowing for normal Treg numbers withproper Teff/Treg ratio. Perhaps, Tregs have a competitive

Fig. 2 Interplay of innate and adaptive immunity. a In the presence ofTregs, IL-2 is primarily utilized by the Tregs rather than by the CD25pos

autoreactive CD4 T cells or NKs, which maintains a proper Teff:Tregbalance and lack of disease progression. b In the absence or lownumber of Tregs such as at diabetes onset or Treg depletion, IL-2 now isprimarily utilized by CD25pos NKs and stimulates IFNγ production,which in turn promotes activation of the adaptive autoimmune immuneresponse tipping the balance toward effector rather than regulatoryresponses


advantage in this microenvironment due to their constitutivelyhigher expression of IL-2Rs over activated CD4/CD8 T cellsor CD25-expressing NKs (Fig. 2a). However, at disease onset,the loss of Tregs could facilitate activation and accumulation ofCD127posCD25pos NKs in the pancreatic microenvironment(Fig. 2b); IFNγ-producing NKs could become activated andexacerbate autoimmunity through enhancement ofautoreactive CD4 and CD8 T-cell effector responses [81,111] or if inordinately high doses of IL-2 are given [112]. Incontrast, a proper LD IL-2 could enhance Tregs and enforcecontrol over these proinflammatory NKs and promote regula-tion, which has not yet been examined. Apart from contribut-ing to an inflammatory environment, NKs responding to IL-2could also compete with IL-2 during diabetes development inNOD mice, thereby impacting immune-based therapies, withor without IL-2. A better understanding of the interrelation-ship between NK subsets and the effect of NKs on pathogenicand regulatory Tcells could help in resolving the controversialrole of NKs in diabetes.

Concluding Remarks

T1D is autoimmune disease that affects millions of peopleworldwide, with a prevalence that has been increasing inmany countries. Approximately 80 people/day are diagnosedin the USA. To date, clinical trials in patients with new onsetT1D have had modest or no clinical efficacy, with overalleffects on preservation of stimulated C-peptide being limitedin magnitude and duration [113]. These outcomes may reflectlimitations of the drugs tested, dosing, timing, duration, androute of administration. In an anti(α)-CD3 trial which targetedT cells, at least a subset of responder patients had better C-peptide preservation than nonresponders, for at least 2 years[114]. In the transplantation setting, the efficacy of pancreaticand islet transplantation in patients with T1D is curtailed bychronic rejection and recurrence of autoimmunity, despite im-munosuppression that prevents rejection [115–117].Additionally, there is a cohort of patients that suffer from viralreactivation and recurrence of autoimmunity, indicative ofchronic latent infections. But again, this immunosuppressionis aimed at targeting T-cell responses. There is an unmet needto develop novel strategies to optimize immune-based thera-pies for the treatment of T1D. The host environment and in-nate immune system are often overlooked while these need tobe understood to develop successful therapies without theneed for chronic immunosuppression. Studies that examinethe role of NKs in disease progression, which NK subsetsimpact the T-cell compartment, and the careful characteriza-tion of the rebounding immune environment followingimmunomodulation are needed in order to develop novelimmune-based therapies. Combination therapies could pro-vide improved outcomes [118–120] but would greatly benefit

with consideration of the impact of NK cells and other com-ponents of the innate immune system.

Compliance with Ethical Standards

Conflict of Interest Chris Fraker and Allison L. Bayer declare that theyhave no conflict of interest.

Human and Animal Rights and Informed Consent This article doesnot contain any studies with human or animal subjects performed by anyof the authors.

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