Page 1 of 63 Diabetes
NKG2D signaling within the pancreatic islets reduces NOD diabetes and increases
protective central memory CD8+ T cell numbers
Andrew P. Trembath1, Kelsey L. Krausz1, Neekun Sharma1, Ivan C. Gerling2, Clayton E. Mathews3 and Mary A. Markiewicz1*
1Department of Microbiology, Molecular Genetics & Immunology, University of Kansas Medical Center, KS City, KS, 66160, USA
2Department of Medicine, University of Tennessee, Memphis, TN, 37996, USA
3Department of Pathology, Immunology and Laboratory Medicine, The University of Florida College of Medicine, Gainesville, FL, 32610, USA
These authors contributed to this work equally
*Corresponding author: Mary A. Markiewicz; [email protected]
Diabetes Publish Ahead of Print, published online June 13, 2020 Diabetes Page 2 of 63
Abstract
NKG2D is implicated in autoimmune diabetes. However, the role of this receptor in diabetes pathogenesis is unclear owing to conflicting results with studies involving global inhibition of
NKG2D signaling. We found that NKG2D and its ligands are present in human pancreata, with expression of NKG2D and its ligands increased in the islets of patients with type 1 diabetes. To directly assess the role of NKG2D in the pancreas, we generated NOD mice that express an
NKG2D ligand in -islet cells. Diabetes was reduced in these mice. The reduction corresponded with a decrease in the effector to central memory CD8+ T cell ratio. Further, NKG2D signaling during in vitro activation of both mouse and human CD8+ T cells resulted in an increased number of central memory CD8+ T cells and diabetes protection by central memory CD8+ T cells in vivo. Taken together, these studies demonstrate that there is a protective role for central memory CD8+ T cells in autoimmune diabetes and that this protection is enhanced with NKG2D signaling. These findings stress the importance of anatomical location when determining the role
NKG2D signaling plays, as well as when developing therapeutic strategies targeting this pathway, in type 1 diabetes development.
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Introduction
Type 1 diabetes is a T cell-mediated autoimmune disorder resulting in the destruction of insulin
producing pancreatic β cells. β cell loss results in an inability of the body to produce insulin.
Despite significant progress in type 1 diabetes research, the immune defects which lead to the
development of this disease remain poorly understood. The immune receptor NKG2D, expressed
by NK cells and subsets of T cells (1-5), is implicated in type 1 diabetes development. However,
its role in disease progression remains unclear, with conflicting reports describing pathogenic (6,
7), non-pathogenic (8), and protective (9) effects of NKG2D signaling.
NKG2D recognizes multiple NKG2D ligands in both humans and mice. These are endogenous
ligands, which are all distantly related to MHC class I in sequence, and are generally believed to
be functionally redundant (10, 11). NKG2D ligands are considered “stress ligands”, with their
expression induced by “cellular stressors”, including viral infection or DNA damage (10, 11).
Engagement of these ligands by NKG2D on NK cells is sufficient to activate NK cell killing of
NKG2D ligand-bearing target cells (1). On T cells, NKG2D engagement generally co-stimulates
TCR-driven activation, enhancing T cell responses (1, 7, 9, 12-14). In addition, NKG2D
signaling has been shown to be important in the development of mouse memory CD8+ T cells
(15-17).
Early studies with the non-obese diabetic (NOD) mouse model led to the hypothesis that
NKG2D signaling, induced by NKG2D ligands expressed on β-islet cells, enhances diabetes
development (6, 7). However, conflicting results were later reported which brought into question
the importance of NKG2D to spontaneous autoimmune diabetes in this pre-clinical model (8, 9).
3 Diabetes Page 4 of 63
Our recent data implicate differential effects of NKG2D signaling induced by microbiota or islet antigen as a source of this controversy (9). Therefore, new experimental approaches are required to determine the various roles of NKG2D in diabetes.
Here we show that mRNAs encoding both NKG2D and NKG2D ligands are expressed in human islets, demonstrating NKG2D signaling in the pancreas is likely relevant to type 1 diabetes pathogenesis. To directly test the effect of NKG2D signaling in the pancreas on autoimmune diabetes, we generated NOD mice with constitutive expression of the mouse NKG2D ligand
RAE1ε on β-islet cells, that is NOD.RIP-RAE1 mice. We found that, despite earlier infiltration of immune cells into the pancreas, these mice developed significantly less diabetes compared to littermate NOD mice. This diabetes reduction corresponded with a reduced ratio of effector and
+ + effector memory CD8 T cells (Teff + em) to central memory CD8 T cells (Tcm). Correlating with these in vivo data, we found that stimulation of NKG2D on NOD and human CD8+ T cells
+ during in vitro activation resulted in a reduced CD8 Teff+em:Tcm ratio. Finally, we found that
+ CD8 Tcm actively suppress NOD diabetes development in vivo. Taken together, these results indicate that NKG2D ligand expression in pancreatic islets protects against autoimmune diabetes
+ development by enhancing the generation or survival of a protective CD8 Tcm population.
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Research Design and Methods
Mice
NOD/ShiLtJ (NOD), NOD.CB17-Prkdcscid/J (NOD.Scid), and C57BL/6 (B6) mice were
purchased from The Jackson Laboratory (stock numbers 001976, 001303, and 000664
respectively). B6.Cg-Tg(Ins2-cre)25Mgn/J (B6.RIP-cre ) mice were purchased from The
Jackson Laboratory (stock #003573). B6.PCCALL-RAE1 and ubiquitous B6.RAE1 mice
were generated previously (7, 18) and provided by Dr. Andrey Shaw (Washington University
School of Medicine). The PCCALL-RAE1 (PCCALL) allele and RIP-Cre transgene were
transferred to the NOD genetic background using speed congenic methods (19) by the
Washington University School of Medicine Mouse Genetics Core. Both NOD.PCCALL and
NOD.RIP-cre strains maintain greater than >98% of the NOD genome, with no alterations in
known Idd loci. Experimental NOD.PCCALL-RAE1, NOD.RIP-cre, and NOD.RIP-RAE1
littermates of both sexes were generated by interbreeding NOD.PCCALL and NOD.RIP-cre
mice. NOD.B6-Klrk1tm1.1Bpol (NOD.Klrk1-/-) mice were previously generated in our laboratory
(9). All mice were housed under specific pathogen-free (SPF) conditions at either the
Washington University School of Medicine or the University of Kansas Medical Center animal
facility in accordance with institutional guidelines and with approval from the Institutional
Animal Care and Use Committee.
Human pancreatic islet microarray
Frozen tissue from cadaveric donors was provided by the Network for Pancreatic Organ Donors
(nPOD) (20) with approval from the University of Florida Health Center Review Board. The
characteristics of the donors used are shown in Table S1. Tissue slides were fixed and laser-
capture of islets conducted as previously described (21). All of the islets in two to five sections
5 Diabetes Page 6 of 63
of tissue from each donor (a minimum of 30 islets each) were captured, pooled, and RNA extracted using the Arcturus PicoPure RNA Isolation Kit (Applied Biosystems, Grand Island,
NY, USA). A Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) was used to assess quality and quantity of RNA. Samples with sufficient RNA quantity and quality were then subjected to gene expression analysis using Affymetrix expression arrays (GeneChip Human
Gene 2.0 ST) and scaled normalized gene expression values produced as previously described
(22).
Insulitis scoring
Insulitis was scored using standard methods (23). The pancreata were fixed in formalin, and
5M sections were generated from the whole tissue and stained with hematoxylin and eosin by
the Washington University School of Medicine Histology Core or the Kansas Intellectual and
Developmental Disabilities Research Center Histology Core. Thirty islets per pancreas in non-
overlapping sections were scored.
Diabetes determination
Diabetes was determined by blood glucose measurements taken weekly via tail vein nick. A
mouse was defined as diabetic on the date when the first of two consecutive blood glucose
measurements ≥250 mg/dL was obtained, as previously described (24).
Flow cytometry
Spleens were dissociated by pressing through a 40um cell strainer in isolation buffer containing
PBS, 2% fetal calf serum, and 2mM EDTA. Pancreata were chopped into small sections,
6 Page 7 of 63 Diabetes
digested with 1mg/ml collagenase IV (Life Technologies) in IMDM for 10 minutes at 37 degrees
with shaking, and a single cell suspension generated by pressing through a 40um cell strainer.
The cells were then washed at least 5 times, followed by antibody staining at 4°C for at least 15
minutes. After staining, cells were washed and analyzed. Cultured cells were washed once in
isolation buffer and stained before similar staining and analysis.
NOD diabetes transfer
Male and female mice were used, with all transfers containing male cells going only into male
NOD.Scid recipients. Spleens and lymph nodes from non-diabetic 6-12 week old NOD mice
were harvested and dissociated by passing through a 40um cell strainer into isolation buffer
containing PBS, 2% fetal calf serum, and 2mM EDTA. CD8+ T cells were then enriched by
negative selection using magnetic bead separation (BD Biosciences Cat. No. 558471) according
to the manufacturer’s instructions. CD8+ T cells were then stained with anti-mCD3-PE-Cy7,
anti-mCD8-APC, anti-mCD44-BV650, and anti-mCD62L-BV510 antibodies (BD Biosciences).
+ + + - + + + + CD3 CD8 CD44 CD62L (Teff + em) and CD3 CD8 CD44 CD62L (Tcm) populations were
separated by fluorescence activated cell sorting by the University of Kansas Medical Center
Flow Core using an AriaIIIu (BD Biosciences). Separately, splenocytes from non-diabetic 6-12
week old mice were depleted of CD8+ T cells by positive selection using anti-mCD8-PE (BD
Biosciences) and anti-PE magnetic bead separation (BD Biosciences Cat. No. 557899). 2.5X106
6 6 6 Teff+em, 2.5X10 Tcm, or a mixture of 2.5X10 Teff + em and 2.5X10 Tcm were each combined with
1.5X107 CD8+ T cell-depleted NOD splenocytes and adoptively transferred into 2-6 month old
NOD.SCID mice by retro-orbital injection.
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In vitro cell culture
All cells were grown in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with
0.1U/mL penicillin, 0.1U/mL streptomycin, 0.29ug/mL L-glutamine, 5.5uM 2-mercaptoethanol, and 10% fetal calf serum. Cells were grown at 37° C at 5% CO2.
In vitro activation of mouse CD8+ T cells
CD8+ T cells were isolated from the spleens and lymph nodes of 6-8 week-old NOD mice by negative selection using magnetic bead separation (BD Biosciences Cat. No. 558471) according to the manufacturer’s instructions. Separately, splenocytes from ubiquitous RAE1ε mice or control C57BL/6 mice were dissociated by passing through a 40um cell strainer and irradiated with 15 Gy using a Cesium-137 irradiator. 106 CD8+ T cells were then plated at with either irradiated C57BL/6 RAE-1ε or C57BL/6 control splenocytes (3X106) along with 1g/mL anti- mCD3 (clone 2C11) in a 24 well plate.
In vitro activation of human CD8+ T cells
Total CD8+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) by negative selection using the Human CD8 T Cell enrichment Set (BD Biosciences), according to manufacturer’s instructions. 24 well plates were incubated with 3μg/mL anti-hCD3 (clone:
OKT3) and anti-hCD28 (clone: 9.3), along with 20μg/mL anti-hNKG2D (clone: 149810), or isotype control in PBS overnight at 4 degrees. The plates were washed with PBS and then the
CD8+ T cells were added to the plate (106 cells/well). After 3 days, cells were split 1:2 and
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maintained in the presence of 20g/mL plate bound anti-hNKG2D. After 5 days of culture, the
cells were harvested for analysis.
Statistical Analyses
Data were collected and analyzed with GraphPad Prism (GraphPad Software). The Log-Rank
(Mantel-Cox) test was used for analysis of diabetes incidence or human NKG2D ligand
expression incidence. Differences between groups were compared using a two-tailed Mann-
Whitney U test unless otherwise indicated. Linear regression analysis was performed to
determine relationships in gene expression in human islets. P values of <0.05 were considered to
be significant and are marked in the figures. Results show mean ± standard error mean unless
otherwise indicated.
Data and Resource Availability
Data Sharing
The primary data generated and analyzed during the current study are either included in the
published article or are available from the corresponding author upon reasonable request.
Resource Sharing
All resources generated during the current study, including all novel mouse strains, are available
from the corresponding author upon reasonable request.
9 Diabetes Page 10 of 63
Results
Increased expression of NKG2D and NKG2D ligands in pancreatic islets of patients with type 1 diabetes
To determine whether studying NKG2D-ligand interaction within the pancreas is relevant to type
1 diabetes, we measured the expression of the mRNAs encoding NKG2D and NKG2D ligands within human pancreatic islets. Pancreatic islets were harvested by laser capture from tissue sections donated to the Network for Pancreatic Organ Donors with Diabetes (nPOD) by people with type 1 diabetes, people with islet antigen-specific autoantibodies but not diabetic (likely pre-diabetic), or non-diabetic age and sex matched controls. Increased transcripts encoding both
NKG2D and NKG2D ligands were detected in pancreatic islets harvested from type 1 diabetes donors, but not individuals positive for islet-specific antibody, compared with non-diabetic donors (Fig. 1). Specifically, the average mean intensity value for the mRNA encoding NKG2D was increased by 20% in the islets from type 1 diabetes patients compared with those from non- diabetic donors (p<0.05 Mann-Whitney U test) (Fig. 1A). Additionally, we found a positive relationship between NKG2D expression and islet T cell infiltration (Fig. S1A). In the case of
NKG2D ligands, 40% of type 1 diabetes donors expressed levels of mRNA encoding at least one ligand at a level determined by Grubb’s test to be an outlier from the values of the controls compared to 5.3% of non-diabetic controls (p<0.009) (Fig. 1B-J). We also found a positive relationship between NKG2D ligand expression and islet T cell infiltration or NKG2D expression (Fig. S1B and C). Together, these data indicate that NKG2D-ligand interactions occur within T cell-infiltrated islets in the pancreas of patients with type 1 diabetes.
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Generation of a novel model to investigate the role of NKG2D signaling within the pancreas
We wished to directly test the effect of NKG2D ligand expression in the pancreas with an animal
model. However, because both NKG2D and NKG2D ligands are expressed on immune cells (25-
28), we could not eliminate these proteins specifically within the pancreas. We therefore tested
this question by increasing NKG2D signaling within the pancreas, which also allowed us to
directly test the hypothesis that NKG2D ligand expression on -islet cells enhances diabetes (6,
7). We moved a cre-inducible RAE1ε transgene we generated previously (PCCALL-RAE1) (7)
and a rat insulin promoter (RIP)-cre transgene onto the NOD background. We then interbred
these mice to generate NOD mice with constitutive expression of RAE1ε in the pancreatic islets
(NOD.RIP-RAE1ε) (Fig. 2A-C).
Engagement of NKG2D by ligand results not only in signaling, but internalization of NKG2D
from the cell surface (29). Therefore, to confirm NKG2D signaling is enhanced in the pancreas
of NOD.RIP-RAE1ε mice as it is in B6.RIP-RAE1ε (7), we compared NKG2D expression on
CD8+ T cells in the pancreas of NOD.RIP-RAE1ε and single transgenic control NOD mice,
NOD.RIP-cre. NKG2D expression was lower on CD8+ T cells in the pancreas of NOD.RIP-
RAE1ε mice compared with NOD.RIP-cre (Fig. 2D). By contrast, NKG2D expression was
similar within the spleen between the two strains (Fig. 2E). These data demonstrate that NKG2D
signaling is enhanced specifically in the pancreas of NOD.RIP-RAE1ε mice.
Diabetes is delayed in RIP-RAE1ε NOD mice via NKG2D signaling
To determine if overexpression of RAE1ε in cells altered NOD islet-specific immunity, we
compared diabetes development between NOD.RIP-RAE1ε (RAE1+ in islets) and control
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NOD.PCCALL (RAE1- in islets) mice. We observed a significant decrease in diabetes in the
NOD.RIP-RAE1ε compared with the NOD.PCCALL littermates (Fig. 3B and C). Due to sexual dimorphism in the NOD model (30-32), a difference in diabetes incidence between male and female mice was expected (Fig. 3B and C). However, in both sexes, RAE1ε expression in the
islet cells significantly reduced diabetes. NKG2D is the only known receptor to interact with
RAE1ε. Therefore, we crossed the RIP-RAE1ε NOD mice to NOD mice genetically deficient in
the gene encoding NKG2D (Klrk1) (9, 33). Diabetes developed similarly in NOD.RIP-
RAE1ε.Klrk1-/- and NOD.PCCALL.Klrk1-/- mice (Fig. 3D). This demonstrates that NKG2D was
required for the reduced diabetes in NOD.RIP-RAE1ε mice.
Earlier insulitis in RIP-RAE1ε NOD mice
To determine the mechanism by which diabetes is reduced in NOD.RIP-RAE1ε mice, we first
compared insulitis development between NOD.RIP-RAE1 and control littermates
(NOD.PCCALL). Consistent with our earlier studies of the B6.RAE1 mice (7), we observed
increased insulitis in the NOD.RIP-RAE1 mice compared with NOD.PCCALL mice at 9 weeks
of age (Fig. 3E). However, at 12 weeks of age, insulitis was similar NOD.PCCALL and
NOD.RIP-RAE1 mice (Fig. 3F). Therefore, we concluded the decrease in diabetes development
in the NOD.RIP-RAE1 mice was not the result of reduced immune infiltration into islets.
+ Reduced CD8 Teff + em:Tcm ratio in RIP-RAE1ε NOD mice
We next compared the immune infiltrate in the pancreas of NOD.RIP-RAE1ε and control
littermates (NOD.PCCALL and NOD.RIP-Cre) at 12 weeks of age, which is a time at which
these strains have similar insulitis and have not yet developed diabetes. We focused on T cells
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and NK cells, as these are the cells on which we detect NKG2D in NOD pancreata and spleens
(9). We found similar numbers of T cells, including CD4+ T and CD8+ T, as well as NK cells, in
the pancreas (Fig. S2A-D) and spleen (Fig. S2E-H) of NOD.RIP-RAE1ε and control littermates
(NOD. PCCALL and NOD.RIP-cre). However, NOD.RIP-RAE1ε mice had a lower percentage
+ hi lo of CD8 T cells with an effector (Teff) or effector memory (Tem) phenotype (CD44 CD62L ) and
+ hi hi a greater percentage of CD8 T cells with a central memory (Tcm) phenotype (CD44 CD62 ) in
the pancreas (Fig. 4A-C, S5B), resulting in a decreased CD8+ Teff + em:Tcm ratio compared to
+ control littermates (NOD.PCCALL and NOD.RIP-cre) (Fig. 4D). The CD8 Teff + em:Tcm ratio
was even lower in the spleen of NOD.RIP-RAE1ε mice (Fig. 4H-K, S5B), and this difference
was consistently observed in both male and female mice (Fig. S3). Demonstrating NKG2D
signaling was required for this effect on CD8+ T cell populations, no difference was observed
between NOD.RIP-RAE1ε-Klrk1-/- versus control littermate NOD.Klrk1-/- (PCCALL/Klrk1-/- and
RIP-cre/Klrk1-/-) (Fig. 4E,F, L, and M). In contrast to these CD8+ T cell population alterations,
+ we did not observe a difference in the CD4 Teff + em:Tcm ratio when comparing NOD.RIP-RAE1
and control littermates (NOD. PCCALL and NOD.RIP-cre) (Fig. 4G, 4N and S4).
+ CD8 Tcm delay NOD diabetes development
+ We hypothesized that the greater number of CD8 Tcm in the NOD.RIP-RAE1 was directly
responsible for the decreased diabetes development in these mice. To test this, we purified CD8+
+ - + + Teff + em (CD44 CD62L ) and Tcm (CD44 CD62L ) populations from non-diabetic NOD mice. We
+ then adoptively transferred Teff + em, Tcm, or Teff + em and Tcm, along with CD8 T cell-depleted NOD
splenocytes, into NOD.Scid recipient mice (Fig. 5A). We measured blood glucose weekly, and
time to diabetes was determined within each experiment relative to the first mouse to become
13 Diabetes Page 14 of 63
diabetic. The mice that received the CD8+ Teff + Tem cells developed diabetes significantly earlier compared with the ones that received Tcm or Tcm+ Teff + em (Fig. 5B). These results demonstrate that
+ NOD CD8 Tcm not only are less diabetogenic compared CD8+ Teff + Tem, but actively suppress
diabetes development.
NKG2D signaling during NOD CD8+ T cell activation results in a reduced Teff+em:Tcm ratio
NKG2D has been shown to be important in CD8+ T cell memory formation in other mouse
+ strains, especially CD8 Tcm (15-17). We therefore hypothesized that NKG2D signaling directly
in NOD CD8+ T cells was responsible for the decreased CD8+ Teff+em:Tcm ratio in the
NOD.RIP-RAE1 mice. To test this, we purified total CD8+ T cells from wild-type NOD mice
and activated them in vitro with splenocytes from B6.RAE1ε, which have ubiquitous transgenic
expression of RAE1ε (18), or control B6 splenocytes. Similar to our observation with CD8+ T
cell populations in vivo in the NOD.RIP-RAE1 mice (Fig. 4), NKG2D stimulation during in
vitro activation with the B6.RAE1 splenocytes resulted in a significantly reduced ratio of Teff +
+ em to Tcm NOD CD8 T cells (Fig. 6).
NKG2D signaling during human CD8+ T cell activation results in a reduced Teff+em:Tcm ratio
We wanted to determine if NKG2D stimulation of human CD8+ T cells similarly decreases the
+ + ratio of Teff + em to Tcm CD8 T cells. To do this, we purified total CD8 T cells from human
peripheral blood and activated these cells in vitro for 5 days in the presence of activating anti-
NKG2D or control antibody. Similar to the mouse CD8+ T cells, we found that NKG2D
+ stimulation during in vitro activation significantly reduced the ratio of human CD8 Teff + em to
Tcm (Fig. 7).
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Discussion
NKG2D and NKG2D ligand expression has been reported in the pancreas of NOD mice (6, 9).
However, the expression of NKG2D and NKG2D ligands in the human pancreas had not been
previously assessed. We demonstrate here that mRNAs encoding NKG2D and NKG2D ligands
are detectable in human pancreatic islets, with increased expression in patients with type 1
diabetes compared with age and sex matched non-diabetic controls, and positively correlates
with T cell infiltration into islets. These findings establish that NKG2D signaling likely occurs in
the pancreas in inflamed islets during type 1 diabetes, supporting continued study of NKG2D
signaling in the pancreas in diabetes pathogenesis.
NKG2D has been implicated in the development of type 1 diabetes. How it affects disease
development, and whether these effects are positive or negative has been a point of uncertainty.
We previously showed a protective effect of NKG2D signaling on CD8+ T cells in NOD
diabetes, but found evidence that NKG2D signaling in the gut may have detrimental effects on
disease development (9). These findings suggest that NKG2D signaling plays different roles in
autoimmune diabetes progression in different anatomical locations. In this study, we endeavored
to more closely assess the role played by NKG2D signaling within the pancreas in autoimmune
diabetes.
Because both NKG2D and ligands are expressed on immune cells (25), we could not eliminate
these proteins specifically within the pancreas. Therefore, to test the effect of NKG2D signaling
in the pancreas, we instead increased NKG2D signaling specifically within the -islet cells of the
pancreas. In our NOD.RIP-RAE1ε model, we selectively increased NKG2D signaling in the
15 Diabetes Page 16 of 63
islets by transgenically expressing RAE1ε using the rat insulin promoter. This expression
resulted in earlier insulitis in the NOD.RIP-RAE1ε mice, which is consistent with our previous
findings with B6.RIP-RAE1ε (7). However, despite this earlier increase, insulitis was similar in
NOD.RIP-RAE1 and control littermates by 12 weeks, and NOD.RIP-RAE1 mice had reduced
+ diabetes development. This diabetes reduction correlated with a decreased ratio of CD8 Teff + em
+ + cells to CD8 Tcm. Given the central role CD8 Teff cells play in autoimmune diabetes pathology
(34, 35), it is not surprising that reduction in these cells correlated with a reduction in diabetes
+ development. In contrast, the increase in CD8 Tcm in NOD.RIP-RAE1ε mice was somewhat
surprising. This increase is unlikely simply a reflection of the lower percentage of NOD.RIP-
RAE1ε mice on their way to developing diabetes. If this was the case, we would have observed a
+ similar increase in CD8 Tcm in male NOD mice, which have a lower diabetes incidence,
compared with female NOD mice, but we did not. Therefore, our data support the hypothesis that
+ this increase in CD8 Tcm does not just correspond to the reduced diabetes in both female and
male NOD.RIP-RAE1ε, but is the cause for the RAE1ε-mediated reduction. This hypothesis is
further supported by the results of the NOD.Scid adoptive cell transfer studies that demonstrated
+ suppression of diabetes development by CD8 Tcm.
+ The cell adoptive transfer studies revealed that CD8 Tcm not only blocked diabetes onset but
+ + also delayed disease transfer by CD8 Teff+em. CD8 Teff + em cells correlate with islet pathology
+ (36), and CD8 Teff cells play a central role in autoimmune diabetes pathology (34, 35). In
+ contrast, CD8 Tcm have reduced cytotoxicity and effector functions (37), correlating with
reduced diabetes transfer by these cells. However, the delay in diabetes we observed with co-
+ + + transfer of CD8 Tcm cells with CD8 Teff + em demonstrates a regulatory effect of the CD8 Tcm
16 Page 17 of 63 Diabetes
+ population. Such a regulatory role for NOD CD8 Tcm was suggested by previous studies
performed by Santamaria and colleagues using transgenic T cell systems (38-40), however this
had not been investigated in parental NOD mice. Tcm can also be precursors to Teff cells (41).
+ + Therefore, the protective cells are likely a subset of the CD8 Tcm population. CD8 Tcm are a
heterogeneous population, which have been shown in other mouse strains to contain a
subpopulation of immune regulatory CD8+ T cells (Tregs) (42). Future studies will need to be
+ performed to determine the identity of the protective NOD CD8 Tcm population, but our results
demonstrate this population is expanded by NKG2D signaling.
Our results indicate that the reduced Teff + em to Tcm ratio in NOD.RIP-RAE1ε mice is due to
enhanced NKG2D signaling directly in CD8+ T cells. We found that NKG2D stimulation during
+ in vitro activation of wild type NOD CD8 T cells drove a similar decrease in the ratio of Teff +
Tem to Tcm as observed in NOD.RIP-RAE1ε mice. This finding that NKG2D stimulation drives a
+ shift towards CD8 Tcm in the NOD mouse is consistent with a role for NKG2D in promoting
memory cell development or survival, particularly Tcm, demonstrated by others with C57BL/6
mice (15-17, 43). Importantly, demonstrating the translatability of the findings of these mouse
studies to the human, we showed that NKG2D signaling during the activation of human CD8+ T
cells also favors Tcm generation over Tem/Teff. Our findings presented here were from studies
performed with total NOD and human CD8+ T cells. In similar studies we performed beginning
with naïve CD8+ T cells, we did not observe this increase in Tcm. This is consistent with the
findings of Wensveen et al. (15), who found that NKG2D signaling in C57BL/6 CD8+ T cells
increased CD8+ Tcm production by promoting the survival of precursor memory cells, rather
than affecting cell proliferation or differentiation. However, additional studies need to be done
17 Diabetes Page 18 of 63
to confirm that the change in CD8+ Teff+em:Tcm ratio in vivo is entirely dependent on NKG2D signaling directly in NOD CD8+ T cells, as well as when during the process of memory cell differentiation NKG2D signaling is important. Further, although our data demonstrate that constitutive high expression of an NKG2D ligand in pancreatic islet cells reduces NOD diabetes and the CD8+ Teff+em:Tcm ratio, it still needs to be determined whether endogenous NKG2D ligand expressed in the pancreas (9) has similar effects.
Initial reports demonstrated that RAE1 family members were expressed by β-islet cells in NOD mice (6), that RAE1 expression by β-islet cells enhanced the recruitment of CD8+ T cells to pancreatic islets (7), and that NKG2D inhibition reduced diabetes in both the NOD and RIP-
LCMV mouse models of autoimmune diabetes, alone or together with CD4+ Tregs, respectively
(6, 44). This led to the hypothesis that inappropriate NKG2D ligand expression by β-islet cells contributed to autoimmune diabetes development or progression. However, we and others have been unable to detect RAE1 expression in the pancreas of NOD mice, and do not detect NKG2D ligand expression by β-islet cells in our NOD colony (9, 45, 46). The reason for these differing reports of RAE1 expression in islets of NOD mice from different mouse colonies is unclear, but may be the result of variation due to genetic drift or differences in microbiota composition.
Regardless, the lack of RAE1 expression in the islets of mice in our NOD colony afforded us a model with which we could directly test the hypothesis that NKG2D ligand expression on β-islet cells promotes NOD diabetes. In contrast to the original hypothesis, our data demonstrate that
RAE1ε expression by islet cells is protective against NOD diabetes and acts via interaction with
NKG2D.
18 Page 19 of 63 Diabetes
Although constitutive exposure to ligand can lead to NKG2D downregulation and dysfunction
(47, 48), our data are consistent with enhanced, not reduced, NKG2D signaling in pancreatic
islets resulting in reduced diabetes in the NOD.RIP-RAE1ε mice. While we observed reduced
NKG2D expression on CD8+ T cells of NOD.RIP-RAE1ε mice in the pancreas, NKG2D
expression by CD8+ T cells in the spleen was unaffected. This is indicative of increased local
NKG2D signaling, as NKG2D is internalized upon ligand engagement and signaling.
Additionally, the reduced diabetes in NOD.RIP-RAE1ε mice correlates with the increased rate of
diabetes in NKG2D-deficient NOD mice we observed with treatment with gut microbiota-
depleting antibiotics in previous studies (9). Finally, our in vitro data demonstrate that NKG2D
+ signaling in CD8 T cells enhances Tcm generation or survival, which correlates with the increase
+ in CD8 Tcm that is observed in NOD.RIP-RAE1 mice. NKG2D is not expressed on mouse
CD8+ T cells until 3-4 days following initial activation (9, 14). This is a time when these cells
start to migrate to the site of antigen (49). Taken together, these data support the conclusion that
NKG2D signaling in islet-specific CD8+ T cells present in the islets during activation enhances
Tcm generation or survival.
In conclusion, we found an increased presence of NKG2D and NKG2D ligand mRNA within the
pancreatic islets of patients with type 1 diabetes and developed a novel mouse model, NOD.RIP-
RAE1ε, to test the effects of NKG2D signaling specifically within the pancreas. We showed that
increased NKG2D signaling within the pancreas of NOD mice had a protective effect, delaying
+ diabetes in NOD.RIP-RAE1ε mice. This correlated with a decrease in the ratio of CD8 Teff +
Tem cells to Tcm cells in NOD.RIP-RAE1ε mice. We demonstrated that NKG2D signaling on
CD8+ T cells drives a similar shift in both human and NOD CD8+ T cell populations in vitro.
19 Diabetes Page 20 of 63
+ + Finally, we found that CD8 Tcm cells transferred significantly less NOD diabetes than CD8 Teff
+ + Tem cells, and had a protective effect, delaying diabetes when co-transferred with CD8 Teff +
Tem cells. We therefore propose a protective role for NKG2D signaling within the pancreas in
+ autoimmune diabetes, by increasing a protective CD8 Tcm population relative to the more
+ pathogenic CD8 Teff + Tem population. These findings reiterate the importance of NKG2D in
autoimmune diabetes, and further stress the importance of anatomical location when determining
the role NKG2D signaling plays in disease development. This is particularly critical when
designing therapeutic strategies that target NKG2D or NKG2D-expressing cells, such as T cells.
Acknowledgements
This research was performed with the support of the Network for Pancreatic Organ donors with
Diabetes (nPOD; RRID:SCR_014641), a collaborative type 1 diabetes research project sponsored
by JDRF (nPOD: 5-SRA-2018-557-Q-R) and The Leona M. & Harry B. Helmsley Charitable Trust
(Grant#2018PG-T1D053). The content and views expressed are the responsibility of the authors
and do not necessarily reflect the official view of nPOD. Organ Procurement Organizations (OPO)
partnering with nPOD to provide research resources are listed at http://www.jdrfnpod.org//for-
partners/npod-partners/.
This work made use of the Flow Cytometry Core Laboratory (supported in part by the
NIH/NIGMS COBRE grant P30 GM103326 and the NIH/NCI Cancer Center Support Grant P30
CA168524), UC4 DK104167 (CEM), UC4 DK104155 (IG & CEM), and the Kansas Intellectual
Developmental Disabilities Research Center Histology Core (supported by NIH U54 HD
090216). Funding for this work was provided by the American Diabetes Association (6-12-JF-
41 to M.A.M.), the JDRF (JDRF 17-2012-595 to C.E.M. & I.C.G.) the American Association of
20 Page 21 of 63 Diabetes
Immunologists (M.A.M. and N.S.), the Washington University School of Medicine Diabetes
Research Center (NIH/NIDDK #P30 DK020579; M.A.M.), the Molecular Regulation of Cell
Development and Differentiation Center of Biomedical Research Excellence (NIH/NIGMS #P30
GM122731; M.A.M.), and the University of Kansas Biomedical Research Training Program
(A.P.T. and K.L.K.). M.A.M. is the guarantor of this work and, as such, had full access to all the
data in the study and takes responsibility for the integrity of the data and the accuracy of the data
analysis.
Author Contributions
A.P.T., K.L.K. and M.A.M. designed and planned the experiments; A.P.T., K.L.K., N.S., I.G.,
and M.A.M. performed the experiments; A.P.T., K.L.K., N.S., C.E.M. and M.A.M. analyzed and
discussed the data; A.P.T. and M.A.M. wrote the manuscript; all authors reviewed the
manuscript.
Declaration of Interests
M.A.M. is a consultant for Johnson & Johnson Global Services. A.P.T., N.S, and C.E.M. declare
no competing financial interests or other conflicts of interest.
21 Diabetes Page 22 of 63
Figure Legends
Figure 1. Increased expression of NKG2D and NKG2D ligand mRNA in pancreatic islets of patients with type 1 diabetes. mRNA expression of the genes encoding (A) NKG2D and (B-I)
NKG2D ligands in the pancreatic islets from individuals with type 1 diabetes (T1D), non- diabetic individuals positive for islet-specific autoantibodies (Ab+), or age and sex matched healthy controls. *p<0.05 in Mann-Whitney U test. (B-I) Data points represent individual samples with colored circles denoting statistical outliers as determined by Grubb’s test. Each color denotes data from one individual. (J) Percent of donors from each group expressing mRNA for at least one NKG2D ligand at a level determined to be an outlier. **p=0.0084 in a Log-Rank test.
Figure 2. Generation of NOD mice with constitutive expression of the NKG2D ligand
RAE1 in the pancreas. (A) Schematic of part of the vector used to generate the PCCALL-
RAE1 mice (7), and (B) breeding schema for generating NOD mice with constitutive RAE1 expression in the islets. Note that rather than RAE1, PCCALL and RIP-Cre mice have constitutive expression of -gal or Cre recombinase in islets, respectively. (C) Flow cytometric analysis of non-immune cells in the pancreas of RIP-Cre or RIP-RAE1 mice stained with an
22 Page 23 of 63 Diabetes
antibody specific for RAE1 (open histogram) or a control antibody (solid histogram). These data
are representative of 3 independent experiments. (D and E) The percentage of CD8+ T cells with
detectable NKG2D expression in the (D) pancreas (n=8) or (E) spleen (n=6) of RIP-RAE1 and
RIP-Cre mice as determined by flow cytometry. *p<0.01 in Mann-Whitney U test.
Figure 3. Diabetes is delayed in RIP-RAE1ε NOD mice through interaction with NKG2D.
(A) Schematic of part of the vector used to generate the PCCALL-RAE1 mice and breeding
schema for generating NOD mice with constitutive RAE1 expression in the islets. (B and C)
Diabetes development in (B) female RIP-RAE1 (n=39) and control (PCCALL) (n=21), and (C)
male RIP-RAE1 (n=23) and control (PCCALL) (n=19), NOD mice. (D) Diabetes development
in female RIP-RAE1 NKG2D-deficient (Klrk1-/-) (n=11) and control (PCCALL/NKG2D-
deficient (Klrk1-/-) (n=20) NOD mice. * p<0.05, **p<0.0008 in Log-Rank test. (E and F)
Insulitis scores in the pancreata of (E) 9 week-old and (F) 12 week-old RIP-RAE1 and control
(RIP-Cre and PCCALL) mice. *p<0.05 in Mann-Whitney U test.
+ Figure 4. Decreased ratio of CD8 Teff + Tem to Tcm in RIP-RAE1ε NOD mice. (A and H)
Representative flow cytometry data gated on CD3+CD8+ cells from the (A) pancreas or (H)
spleen of a RIP-RAE1 (top panel) or control (bottom panel) mouse showing CD44 and CD62L
expression. The numbers shown are the percentage of cells present in each quadrant of the dot
+ + + - plot. (B and I) The percentage of CD8 CD3 cells that were CD44 CD62L (Teff + Tem),
+ + - - CD44 CD62L (Tcm), or CD44 CD62L (naïve) in the (B) pancreas or (I) spleen of RIP-RAE1ε
mice (n=22) and control (RIP-Cre and PCCALL) mice (n=19). (C and J) The number of cells
+ - + + - - that were CD44 CD62L (Teff + Tem), CD44 CD62L (Tcm), or CD44 CD62L (naïve) in the (C)
23 Diabetes Page 24 of 63
pancreas and (J) spleen of RIP-RAE1ε mice (n=22) and control (RIP-Cre and PCCALL) mice
+ (n=19). (D and K) The ratio of CD8 Teff + Tem:Tcm in the (D) pancreata and (K) spleen of RIP-
RAE1 and control (RIP-Cre and PCCALL) mice. (E and L) The ratio of CD8+ Teff + Tem:Tcm in the (E) pancreata and (L) spleen of RIP-RAE1 and control (RIP-Cre and PCCALL) Klrk1-/-
+ - + + mice. (F and M) The percentage of cells that were CD44 CD62L (Teff + Tem), CD44 CD62L
- - -/- (Tcm), or CD44 CD62L (naïve) in the (F) pancreas and (M) spleen of RIP-RAE1ε/ Klrk1
(n=15) and control (RIP-Cre and PCCALL)/Klrk1-/- (n=20) mice. (G and N). The ratio of CD4+
Teff + Tem:Tcm in the (G) pancreata and (M) spleen of RIP-RAE1 (n=22) and control (RIP-Cre
and PCCALL) (n=19) mice. *p<0.05, **p<0.01 in a two-sided Mann-Whitney U test.
Figure 5. CD8+ Tcm delay NOD diabetes development. (A) Schematic of adoptive transfer
experimental design. M: million. (B) The number of days to diabetes in NOD.Scid recipient
mice. The day the first mouse in each experiment developed diabetes was called “day 0”. The
data shown are combined data from five independent experiments. Open points denote mice that
were did not develop diabetes within 30 weeks after adoptive transfer. *p< 0.05 and ***p< 0.001
in Log-Rank test.
Figure 6. NKG2D signaling in NOD CD8+ T cells increases CD8+ Tcm. (A) Representative
flow cytometry data gated on in vitro activated NOD CD3+CD8+ cells showing CD44 and
CD62L expression. The numbers shown are the percentage of cells present in each quadrant of
the dot plot. (B and C) The percentage (mean +/- STD) of NOD CD8+CD3+ cells that were (B)
+ + + - CD44 CD62L (Tcm) or (C) CD44 CD62L (Teff + em) after activation in vitro in the presence of
RAE1+ or RAE1- splenocytes. (D) The ratio (mean +/- STD) of NOD CD8+ Teff + em:Tcm
24 Page 25 of 63 Diabetes
generated after activation in vitro in the presence of RAE1+ or RAE1- splenocytes. These data
are representative of 3 independent experiments. ****p<0.0001 in two-sided t-test.
Figure 7. NKG2D signaling in human CD8+ T cells increases CD8+ Tcm. (A and C)
Representative flow cytometry data gated on in vitro-activated human total CD3+CD8+ cells
showing CCR7 and CD45RO expression. The numbers shown are the percentage of cells present
+ + - in each quadrant of the dot plot. (B) The ratio of human CD8 Teff+em (CD45RO CCR7 ):Tcm
(CD45RO+CCR7+) generated after activation of human total CD3+CD8+ cells in vitro in the
presence or absence of an activating anti-NKG2D antibody. These data are the combined results
of five independent experiments. *p<0.05 in one-tailed Mann-Whitney test.
25 Diabetes Page 26 of 63
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Figure 1. Increased expression of NKG2D and NKG2D ligand mRNA in pancreatic islets of patients with type 1 diabetes. Diabetes Page 30 of 63
Figure 2. Generation of NOD mice with constitutive expression of the NKG2D ligand RAE1e in the pancreas. Page 31 of 63 Diabetes
Figure 3. Diabetes is delayed in RIP-RAE1ε NOD mice through interaction with NKG2D. Diabetes Page 32 of 63
Figure 4. Decreased ratio of CD8+ Teff + Tem to Tcm in RIP-RAE1ε NOD mice. Page 33 of 63 Diabetes
Figure 5. CD8+ Tcm delay NOD diabetes development. Diabetes Page 34 of 63
Figure 6. NKG2D signaling in NOD CD8+ T cells increases CD8+ Tcm. Page 35 of 63 Diabetes
Figure 7. NKG2D signaling in human CD8+ T cells increases CD8+ Tcm. Diabetes Page 36 of 63
Supplementary Table 1. Pancreatic organ donor characteristics. Sample ID Clinical Age Sex Race BMI HLA Risk Diabetes phenotype (years) duration (years) 6013 Control 65 M Caucasian 24.2 Neutral 0 6024 Control 21 M Caucasian 27.8 Predisposition 0 6048 Control 30 M Caucasian 20.6 Protective 0 6075 Control 16 M African 14.9 Neutral 0 American 6012 Control 68 F Caucasian 23.7 Protective 0 6099 Control 14.2 M Caucasian 30 Predisposition 0 6140 Control 38 M Caucasian 21.7 Predisposition 0 6162 Control 22.7 M African 28.9 Neutral 0 American 6168 Control 51 M Hispanic/Latino 25.2 Predisposition 0 6227 Control 17 F Caucasian 26.4 Predisposition 0 6129 Control 42.9 F Caucasian 23.4 Protective 0 6165 Control 45.8 F Caucasian 25 Protective 0 6102 Control 45.1 F Caucasian 35.1 Predisposition 0 6229 Control 31 F Caucasian 26.9 Neutral 0 6251 Control 33 F Caucasian 29.5 Neutral 0 6010 Control 47 F Caucasian 19.7 Protective 0 6179 Control 21.8 F Caucasian 20.7 Predisposition 0 6019 Control 42 M Caucasian 31 Predisposition 0 6080 Antibody+ 69.2 F Caucasian 21.3 Neutral 0 6123 Antibody+ 23.2 F Caucasian 17.6 Neutral 0 6158 Antibody+ 40.3 M Caucasian 29.7 Neutral 0 6167 Antibody+ 37 M Caucasian 26.3 Predisposition 0 6171 Antibody+ 4.3 F Caucasian 14.8 Predisposition 0 6044 Antibody+ 41.4 M Hispanic/Latino 27.4 Protective 0 6101 Antibody+ 64.8 M Caucasian 34.3 Protective 0 6154 Antibody+ 48.5 F Caucasian 24.5 Protective 0 6156 Antibody+ 40 M Caucasian 19.8 Neutral 0 6181 Antibody+ 31.9 M Caucasian 21.9 Predisposition 0 6197 Antibody+ 22 M African 28.2 Neutral 0 American 6147 Antibody+ 23.8 F Caucasian 32.9 Neutral 0 6070 T1D 22.6 F Caucasian 21.6 Neutral 7 Page 37 of 63 Diabetes
6084 T1D 14.2 M Caucasian 26.3 Predisposition 4 6088 T1D 31.2 M Caucasian 27 Predisposition 5 6180 T1D 27.1 M Caucasian 25.9 Predisposition 11 6224 T1D 21 F Caucasian 22.8 Predisposition 1.5 6243 T1D 13 M Caucasian 21.3 Predisposition 5 6228 T1D 13 M Caucasian 17.4 Predisposition 0 6209 T1D 5 F Caucasian 11.95 Predisposition 0.25 6038 T1D 37.2 F Caucasian 30.9 Predisposition 20 6046 T1D 18.8 F Caucasian 25.2 Predisposition 8 6069 T1D 22.9 M African 28.8 Predisposition 7 American 6195 T1D 19.2 M Caucasian 23.7 Neutral 5 6052 T1D 12 M African 20.3 Neutral 1 American 6268 T1D 12 F Caucasian 26.6 Predisposition 3 6265 T1D 11 M Caucasian 12.9 Predisposition 8 6196 T1D 26 F African 26.6 Neutral 15 American 6211 T1D 24 F African 24.4 Predisposition 4 American 6113 T1D 13.1 F Caucasian 24.8 Predisposition 1.58 6264 T1D 12 F Caucasian 22 Predisposition 9 6235 T1D 43.5 M Caucasian 28.7 Neutral 21 Diabetes Page 38 of 63
A B R2= 0.12 R2=0.13 p=0.0138 p=0.0091 Klrk1 RAET1L
CD3e CD3e
C R2=0.12 p=0.0127 RAET1L
Klrk1 Supplementary Figure 1. Klrk1 expression correlates with CD3e, and RAET1L expression in human pancreatic islets. Linear regression analysis demonstrating a positive correlation between expression of (A) Klrk1 and CD3e, (B) RATE1L and CD3e, and (C) RAET1L and Klrk1 in human pancreatic islets. n=50 (18 controls, 12 Ab+, 20 T1D) Page 39 of 63 Diabetes
Pancreas
Control NK Cells Total T cells RIP-RAE1ε A B NS NS %Live Cells %Live %Live Cells %Live
Control RIP-RAE1ε Control RIP-RAE1ε
NS C NS D Cell Count Cell Cell Count Cell
Control RIP-RAE1ε Control RIP-RAE1ε
Spleen
NK Cells Total T cells Control E F NS NS RIP-RAE1ε %Live Cells %Live %Live Cells %Live
Control RIP-RAE1ε Control RIP-RAE1ε
G NS H NS
Control RIP-RAE1ε Control RIP-RAE1ε
Supplementary Figure 2. RIP-RAE1e and control mice have similar numbers of T and NK cells in the pancreata and spleens. (A, C, E and G) The (A and E) percentage or (C and G) number of NK cells in the (A and C) pancreas or (E and G) spleen of RIP-RAE1e (n=22) and control (RIP-Cre and PCCALL) (n=19) mice. (B, D, F and H) The (B and F) percentage or (D and G) number of T cells in the (B and D) pancreas or (F and H) spleen of RIP-RAE1e (n=22) and control (RIP-Cre and PCCALL) (n=19) mice. Supplementary Figure Supplementary Whitney U Test. (G) RIP female CD3+CD8+ T cells that (B) ofcells CD3+CD8+ that were CD44hiCD62Llo (C) (C) of spleen female RIP female male or A C E G
- + + RAE1
(H) %CD3 CD8 Ratio Teff + em : Tcm Ratio Teff + em : Tcm %CD3+CD8+ male or (T - CD62L (T CD44 RAE1 eff female RIP female CD62L RIP CD44 eff e + T RIP * + T and control ( * - hi em Cre lo (D) (D) e - hi em Cre ) lo and control (RIP were CD44hiCD62Llo 3. Decreased CD8+ CD62L Males Males ) Males Males CD44 female female (T *** CD62L CD44 ** (T - cm RAE1 RIP cm ) hi RIP hi ) hi hi RIP - RIP RAE1 CD62L (naïve) CD44 - e RAE1 CD62L (naïve) CD44 - and control (RIP - Cre RAE1 ε lo hi ε - lo Cre and PCCALL) mice. hi e Teff+em:Tcm and PCCALL and control (RIP , CD44hiCD62Lhi , CD44hiCD62Lhi, or CD44loCD62Lhi or , CD44hiCD62Lhi, Spleen Pancreas - Cre Diabetes and PCCALL) mice. *p<,0.05 **p<0.01, ***p<0.001 - Mann ) mice. ) mice. ratio in RIP D B F - H (G and H) and (G Cre + + Ratio Teff + em : Tcm
, or CD44loCD62Lhi, or %CD3 CD8 + +
( Ratio Teff + em : Tcm %CD3 CD8 and PCCALL) mice. C and D) (T CD62L (T CD44 eff CD62L CD44 RIP eff RIP + T - * + T RAE1 The CD8+ - hi - em Cre lo Cre hi em Females Females lo ) Females Females The CD8+ CD62L ) CD44 CD62L (T e CD44
* (T * NOD NOD mice. cm * RIP cm RIP ) hi hi ) Teff+em:Tcm hi hi - - RAE1 in the spleen of spleen in the CD62L RAE1 in the pancreas of pancreas in the (naïve) CD44 (E and F) F) and (E CD62L Teff+em:Tcm (naïve) CD44 ε ε lo hi (A B and lo hi RIP Control RIP Control The percentage of ratio in the spleen of of spleen the in ratio - - RAE1 RAE1 (E) ratio in the the in ratio ) The percentage percentage ) The ε ε (A) male or male or Page 40of63 (F) Page 41 of 63 Diabetes Pancreas CD4+ T Cells A B Control * RIP-RAE1ε + CD4 + Cell Number Cell %CD3
Teff + Tem Tcm naïve Teff + Tem Tcm naïve Males Female C D + + CD4 CD4 + + %CD3 %CD3
Teff + Tem Tcm naïve Teff + Tem Tcm naïve Spleen CD4+ T Cells
E F Control RIP-RAE1ε + CD4 + Cell Number Cell %CD3
Teff + Tem Tcm naïve Teff + Tem Tcm naïve
G Males H Female + + CD4 CD4 + + %CD3 %CD3
Teff + Tem Tcm naïve Teff + Tem Tcm naïve
Supplementary Figure 4. Similar ratio of CD4+ Teff:Tem:Tcm in RIP-RAE1e and control NOD mice. (A, C, and D) The percentage of CD3+CD4+ cells that were CD44hiCD62Llo, CD44hiCD6L2hi, or CD44loCD62Lhi in the pancreas of (A) all RIP-RAE1e and control mice, (C) male RIP-RAE1e and control, or (D) female RIP-RAE1e and control mice. (B) The number of CD3+CD4+ cells that were CD44hiCD62Llo, CD44hiCD62Lhi, or CD44loCD62Lhi in the pancreas of RIP-RAE1e and control mice. (E, G, and H) The percentage of CD3+CD4+ cells that were CD44hiCD62Llo, CD44hiCD62Lhi, or CD44loCD62Lhi in the spleen of (E) all RIP-RAE1e and control mice, (G) male RIP-RAE1e and control, or (H) female RIP-RAE1e and control mice. (F) The number of CD3+CD4+ cells that were CD44hiCD62Llo, CD44hiCD62Lhi, or CD44loCD62Lhi in the spleen of RIP-RAE1e and control (RIP-Cre and PCCALL) mice. *p<0.05, **p<0.01 Mann-Whitney U Test. Diabetes Page 42 of 63
A Gating strategy for human CD8+ T cells
B NOD CD8+ T cells
Supplementary Figure 5. Flow Cytometry Gating Strategies. Gating strategy for (A) human or (B) mouse CD8+ T cells . Page 43 of 63 Diabetes
Checklist for Reporting Human Islet Preparations Used in Research
Adapted from Hart NJ, Powers AC (2018) Progress, challenges, and suggestions for using human islets to understand islet biology and human diabetes. Diabetologia https://doi.org/10.1007/s00125-018-4772-2.
Manuscript DOI: https://doi.org/10.2337/DB19-0979.R1 Title: NKG2D signaling within the pancreatic islets reduces NOD diabetes and increases protective central memory CD8+ T cell numbers Author list: Andrew P. Trembath, Kelsey L. Krausz, Neekun Sharma, Ivan C. Gerling, Clayton E. Mathews and Mary A. Markiewicz
Corresponding author: Mary A. Markiewicz Email address: [email protected]
Islet preparation 1 2 3 4 5 6 7 8
MANDATORY INFORMATION
Unique identifier 6013 6024 6048 6075 6012 6099 6140 6162
Donor age (years) 65 21 30 16 68 14.2 38 22.7
Donor sex (M/F) M M M M F M M M
Donor BMI (kg/m2) 24.2 27.8 20.6 14.9 23.7 30 21.7 28.9 Diabetes Page 44 of 63
Donor HbA1c or other measure of blood glucose 0 0 0 0 0 0 6 0 control
Origin/source of isletsb nPOD nPOD nPOD nPOD nPOD nPOD nPOD nPOD
University of University of University of University of University of University of University of University of Islet isolation centre Florida Florida Florida Florida Florida Florida Florida Florida
Donor history of No No No No No No No No diabetes? Yes/No
If Yes, complete the next two lines if this information is available
Diabetes duration (years)
Glucose-lowering therapy at time of deathc
RECOMMENDED INFORMATION
Donor cause of Cerebrovascular/ Head Cerebrovascular/ Cerebrovascular/ Head Cerebrovascular/ Head Anoxia death Stroke Trauma Stroke Stroke Trauma Stroke Trauma
Warm ischaemia Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown time (h)
Cold ischaemia Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown time (h)
Version 1.0, created 16 Nov 2018 Page 45 of 63 Diabetes
Estimated purity N/A N/A N/A N/A N/A N/A N/A N/A (%)
Estimated viability N/A N/A N/A N/A N/A N/A N/A N/A (%)
Total culture time N/A N/A N/A N/A N/A N/A N/A N/A (h)d Glucose-stimulated insulin secretion or N/A N/A N/A N/A N/A N/A N/A N/A other functional measuremente Handpicked to No No No No No No No No purity? Yes/No Additional notes Islets isolated by Islets Islets isolated by Islets Islets isolated by Islets Islets isolated by Islets laser capture isolated laser capture isolated laser capture isolated laser capture isolated from tissue by laser from tissue by laser from tissue by laser from tissue by laser sections capture sections capture sections capture sections capture from from from from tissue tissue tissue tissue sections sections sections sections
aIf you have used more than eight islet preparations, please complete additional forms as necessary bFor example, IIDP, ECIT, Alberta IsletCore cPlease specify the therapy/therapies dTime of islet culture at the isolation centre, during shipment and at the receiving laboratory ePlease specify the test and the results
Version 1.0, created 16 Nov 2018 Diabetes Page 46 of 63
Checklist for Reporting Human Islet Preparations Used in Research
Adapted from Hart NJ, Powers AC (2018) Progress, challenges, and suggestions for using human islets to understand islet biology and human diabetes. Diabetologia https://doi.org/10.1007/s00125-018-4772-2.
Manuscript DOI: https://doi.org/10.2337/DB19-0979.R1
Title: NKG2D signaling within the pancreatic islets reduces NOD diabetes and increases protective central memory CD8+ T cell numbers
Author list: Andrew P. Trembath, Kelsey L. Krausz, Neekun Sharma, Ivan C. Gerling, Clayton E. Mathews and Mary A. Markiewicz
Corresponding author: Mary A. Markiewicz Email address: [email protected]
Islet preparation 9 10 11 12 13 14 15 16
MANDATORY INFORMATION
Unique identifier 6168 6227 6129 6165 6102 6229 6251 6010
Donor age (years) 51 17 42.9 45.8 45.1 31 33 47
Donor sex (M/F) M F F F F F F F
Donor BMI (kg/m2) 25.2 26.4 23.4 25 35.1 26.9 29.5 19.7 Page 47 of 63 Diabetes
Donor HbA1c or other measure of blood glucose 6.2 0 5.2 5.6 6.1 5.5 5.3 0 control
Origin/source of isletsb nPOD nPOD nPOD nPOD nPOD nPOD nPOD nPOD
University of University of University of University of University of University of University of University of Islet isolation centre Florida Florida Florida Florida Florida Florida Florida Florida
Donor history of No No No No No No No No diabetes? Yes/No
If Yes, complete the next two lines if this information is available
Diabetes duration (years)
Glucose-lowering therapy
at time of deathc
RECOMMENDED INFORMATION
Donor cause of Cerebrovascular/ Cerebrovascular/ Cerebrovascular/ Cerebrovascular/ Head Head Cerebrovascular/ Anoxia death Stroke Stroke Stroke Stroke Trauma Trauma Stroke
Warm ischaemia Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown time (h)
Cold ischaemia Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown time (h)
Version 1.0, created 16 Nov 2018 Diabetes Page 48 of 63
Estimated purity N/A N/A N/A N/A N/A N/A N/A N/A (%)
Estimated N/A N/A N/A N/A N/A N/A N/A N/A viability (%)
Total culture N/A N/A N/A N/A N/A N/A N/A N/A time (h)d Glucose- stimulated insulin secretion N/A N/A N/A N/A N/A N/A N/A N/A or other functional measuremente Handpicked to No No No No No No No No purity? Yes/No Additional notes Islets isolated by Islets isolated by Islets Islets isolated by Islets isolated by Islets Islets Islets isolated by laser capture laser capture isolated laser capture laser capture isolated isolated laser capture from tissue from tissue by laser from tissue from tissue by laser by laser from tissue sections sections capture sections sections capture capture sections from from from tissue tissue tissue sections sections sections aIf you have used more than eight islet preparations, please complete additional forms as necessary bFor example, IIDP, ECIT, Alberta IsletCore cPlease specify the therapy/therapies dTime of islet culture at the isolation centre, during shipment and at the receiving laboratory ePlease specify the test and the results
Version 1.0, created 16 Nov 2018 Page 49 of 63 Diabetes
Checklist for Reporting Human Islet Preparations Used in Research
Adapted from Hart NJ, Powers AC (2018) Progress, challenges, and suggestions for using human islets to understand islet biology and human diabetes. Diabetologia https://doi.org/10.1007/s00125-018-4772-2.
Manuscript DOI: https://doi.org/10.2337DB19-0979.R1
Title: NKG2D signaling within the pancreatic islets reduces NOD diabetes and increases protective central memory CD8+ T cell numbers
Author list: Andrew P. Trembath, Kelsey L. Krausz, Neekun Sharma, Ivan C. Gerling, Clayton E. Mathews and Mary A. Markiewicz
Corresponding author: Mary A. Markiewicz Email address: [email protected]
Islet preparation 17 18 19 20 21 22 23 24
MANDATORY INFORMATION
Unique identifier 6179 6019 6070 6084 6088 6180 6224 6243
Donor age (years) 21.8 42 22.6 14.2 31.2 27.1 21 13
Donor sex (M/F) F M F M M M F M
Donor BMI (kg/m2) 20.7 31 21.6 26.3 27 25.9 22.8 21.3 Diabetes Page 50 of 63
Donor HbA1c or other measure of blood glucose 0 5.6 0 0 0 0 0 13.1 control
Origin/source of isletsb nPOD nPOD nPOD nPOD nPOD nPOD nPOD nPOD
University of University of University of University of University of University of University of University of Islet isolation centre Florida Florida Florida Florida Florida Florida Florida Florida
Donor history of No No Yes Yes Yes Yes Yes Yes diabetes? Yes/No
If Yes, complete the next two lines if this information is available
Diabetes duration (years) 7 4 5 11 1.5 5
Glucose-lowering therapy Unknown Unknown Unknown Unknown Unknown Unknown at time of deathc
RECOMMENDED INFORMATION
Head Head Head Head Cerebrovascular/ Donor cause of death Anoxia Anoxia Anoxia Trauma Trauma Trauma Trauma Stroke
Warm ischaemia time Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown (h)
Cold ischaemia time (h) Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown
Version 1.0, created 16 Nov 2018 Page 51 of 63 Diabetes
Estimated purity (%) N/A N/A N/A N/A N/A N/A N/A N/A
Estimated viability (%) N/A N/A N/A N/A N/A N/A N/A N/A
Total culture time (h)d N/A N/A N/A N/A N/A N/A N/A N/A
Glucose-stimulated insulin secretion or other N/A N/A N/A N/A N/A N/A N/A N/A functional measuremente Handpicked to purity? No No No No No No No No Yes/No Additional notes Islets Islets Islets Islets Islets Islets Islets Islets isolated by isolated by isolated by isolated by isolated by isolated by isolated by isolated by laser capture laser laser laser laser laser laser laser from tissue capture from capture from capture from capture from capture from capture from capture from sections tissue tissue tissue tissue tissue tissue tissue sections sections sections sections sections sections sections
aIf you have used more than eight islet preparations, please complete additional forms as necessary bFor example, IIDP, ECIT, Alberta IsletCore cPlease specify the therapy/therapies dTime of islet culture at the isolation centre, during shipment and at the receiving laboratory ePlease specify the test and the results
Version 1.0, created 16 Nov 2018 Diabetes Page 52 of 63
Checklist for Reporting Human Islet Preparations Used in Research
Adapted from Hart NJ, Powers AC (2018) Progress, challenges, and suggestions for using human islets to understand islet biology and human diabetes. Diabetologia https://doi.org/10.1007/s00125-018-4772-2.
Manuscript DOI: https://doi.org/10.2337/DB19-0979.R1
Title: NKG2D signaling within the pancreatic islets reduces NOD diabetes and increases protective central memory CD8+ T cell numbers
Author list: Andrew P. Trembath, Kelsey L. Krausz, Neekun Sharma, Ivan C. Gerling, Clayton E. Mathews and Mary A. Markiewicz
Corresponding author: Mary A. Markiewicz Email address: [email protected]
Islet preparation 25 26 27 28 29 30 31 32
MANDATORY INFORMATION
Unique identifier 6228 6209 6038 6046 6069 6195 6052 6268
Donor age (years) 13 5 37.2 18.8 22.9 19.2 12 12
Donor sex (M/F) M F F F M M M F
Donor BMI (kg/m2) 17.4 11.95 30.9 25.2 28.2 23.7 20.3 26.6 Page 53 of 63 Diabetes
1 3 1 1 2 2 5 1
Donor HbA1c or other measure of blood glucose 13.3 0 0 0 0 0 0 9.8 control
Origin/source of isletsb nPOD nPOD nPOD nPOD nPOD nPOD nPOD nPOD
University of University of University of University of University of University of University of University of Islet isolation centre Florida Florida Florida Florida Florida Florida Florida Florida
Donor history of Yes Yes Yes Yes Yes Yes Yes Yes diabetes? Yes/No
If Yes, complete the next two lines if this information is available
Diabetes duration (years) 0 0.25 20 8 7 5 1 3
Glucose-lowering therapy Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown at time of deathc
RECOMMENDED INFORMATION
Head Head Donor cause of death Anoxia DKA Anoxia Anoxia Another Anoxia Trauma Trauma
Warm ischaemia time (h) Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown
Cold ischaemia time (h) Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown
Version 1.0, created 16 Nov 2018 Diabetes Page 54 of 63
1 3 1 1 2 2 5 1
Estimated purity (%) N/A N/A N/A N/A N/A N/A N/A N/A
Estimated viability (%) N/A N/A N/A N/A N/A N/A N/A N/A
Total culture time (h)d N/A N/A N/A N/A N/A N/A N/A N/A
Glucose-stimulated insulin secretion or other N/A N/A N/A N/A N/A N/A N/A N/A functional measuremente Handpicked to purity? No No No No No No No No Yes/No Additional notes Islets Islets Islets Islets Islets Islets Islets Islets isolated by isolated by isolated by isolated by isolated by isolated by isolated by isolated by laser capture laser capture laser capture laser capture laser capture laser capture laser capture laser capture from tissue from tissue from tissue from tissue from tissue from tissue from tissue from tissue sections sections sections sections sections sections sections sections aIf you have used more than eight islet preparations, please complete additional forms as necessary bFor example, IIDP, ECIT, Alberta IsletCore cPlease specify the therapy/therapies dTime of islet culture at the isolation centre, during shipment and at the receiving laboratory ePlease specify the test and the results
Version 1.0, created 16 Nov 2018 Page 55 of 63 Diabetes
Checklist for Reporting Human Islet Preparations Used in Research
Adapted from Hart NJ, Powers AC (2018) Progress, challenges, and suggestions for using human islets to understand islet biology and human diabetes. Diabetologia https://doi.org/10.1007/s00125-018-4772-2.
Manuscript DOI: https://doi.org/10.2337/DB19-0979.R1
Title: NKG2D signaling within the pancreatic islets reduces NOD diabetes and increases protective central memory CD8+ T cell numbers
Author list: Andrew P. Trembath, Kelsey L. Krausz, Neekun Sharma, Ivan C. Gerling, Clayton E. Mathews and Mary A. Markiewicz
Corresponding author: Mary A. Markiewicz Email address: [email protected]
Islet preparation 33 34 35 36 37 38 39 40
MANDATORY INFORMATION
Unique identifier 6265 6196 6211 6113 6264 6135 6080 6123
Donor age (years) 11 26 24 13.1 12 43.5 69.2 23.2
Donor sex (M/F) M F F F F M F F
Donor BMI (kg/m2) 12.9 26.6 24.4 24.8 22 28.7 21.3 17.6 Diabetes Page 56 of 63
Donor HbA1c or other measure of blood glucose 0 0 10.5 0 8.9 0 0 5.4 control
Origin/source of isletsb nPOD nPOD nPOD nPOD nPOD nPOD nPOD nPOD
University of University of University of University of University of University of University of University of Islet isolation centre Florida Florida Florida Florida Florida Florida Florida Florida
Donor history of Yes Yes Yes Yes Yes Yes No No diabetes? Yes/No
If Yes, complete the next two lines if this information is available
Diabetes duration (years) 8 15 4 1.58 9 21 0 0
Glucose-lowering therapy Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown at time of deathc
RECOMMENDED INFORMATION
Cerebrovascular/ Head Cerebrovascular/ Head Donor cause of death Anoxia Anoxia DKA Anoxia Stroke Trauma Stroke Trauma
Warm ischaemia time Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown (h)
Cold ischaemia time (h) Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown
Version 1.0, created 16 Nov 2018 Page 57 of 63 Diabetes
Estimated purity (%) N/A N/A N/A N/A N/A N/A N/A N/A
Estimated viability (%) N/A N/A N/A N/A N/A N/A N/A N/A
Total culture time (h)d N/A N/A N/A N/A N/A N/A N/A N/A
Glucose-stimulated insulin secretion or N/A N/A N/A N/A N/A N/A N/A N/A other functional measuremente Handpicked to purity? No No No No No No No No Yes/No Additional notes Islets isolated by Islets Islets Islets Islets Islets Islets isolated by Islets laser capture isolated by isolated by isolated by isolated by isolated by laser capture isolated by from tissue laser laser laser laser laser from tissue laser sections capture capture capture capture capture sections capture from tissue from tissue from tissue from tissue from tissue from tissue sections sections sections sections sections sections
aIf you have used more than eight islet preparations, please complete additional forms as necessary bFor example, IIDP, ECIT, Alberta IsletCore cPlease specify the therapy/therapies dTime of islet culture at the isolation centre, during shipment and at the receiving laboratory ePlease specify the test and the results
Version 1.0, created 16 Nov 2018 Diabetes Page 58 of 63
Checklist for Reporting Human Islet Preparations Used in Research
Adapted from Hart NJ, Powers AC (2018) Progress, challenges, and suggestions for using human islets to understand islet biology and human diabetes. Diabetologia https://doi.org/10.1007/s00125-018-4772-2.
Manuscript DOI: https://doi.org/10.2337/DB19-0979.R1
Title: NKG2D signaling within the pancreatic islets reduces NOD diabetes and increases protective central memory CD8+ T cell numbers
Author list: Andrew P. Trembath, Kelsey L. Krausz, Neekun Sharma, Ivan C. Gerling, Clayton E. Mathews and Mary A. Markiewicz
Corresponding author: Mary A. Markiewicz Email address: [email protected]
Islet preparation 41 42 43 44 45 46 47 48
MANDATORY INFORMATION
Unique identifier 6158 6167 6171 6044 6101 6154 6156 6181
Donor age (years) 40.3 37 4.3 41.4 64.8 48.5 40 31.9
Donor sex (M/F) M M F M M F M M
Donor BMI (kg/m2) 29.7 26.3 14.8 27.4 34.3 24.5 19.8 21.9 Page 59 of 63 Diabetes
Donor HbA1c or other measure of blood glucose 5.6 0 0 0 0 0 0 0 control
Origin/source of isletsb nPOD nPOD nPOD nPOD nPOD nPOD nPOD nPOD
University of University of University of University of University of University of University of University of Islet isolation centre Florida Florida Florida Florida Florida Florida Florida Florida
Donor history of No No No No No No No No diabetes? Yes/No
If Yes, complete the next two lines if this information is available
Diabetes duration (years)
Glucose-lowering therapy
at time of deathc
RECOMMENDED INFORMATION
Head Head Head Cerebrovascular/ Head Head Head Donor cause of death Anoxia Trauma Trauma Trauma Stroke Trauma Trauma Trauma
Warm ischaemia time Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown (h)
Cold ischaemia time (h) Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown
Version 1.0, created 16 Nov 2018 Diabetes Page 60 of 63
Estimated purity (%) N/A N/A N/A N/A N/A N/A N/A N/A
Estimated viability (%) N/A N/A N/A N/A N/A N/A N/A N/A
Total culture time (h)d N/A N/A N/A N/A N/A N/A N/A N/A
Glucose-stimulated insulin secretion or other N/A N/A N/A N/A N/A N/A N/A N/A functional measuremente Handpicked to purity? No No No No No No No No Yes/No Additional notes Islets Islets Islets Islets Islets isolated by Islets Islets Islets isolated by isolated by isolated by isolated by laser capture isolated by isolated by isolated by laser laser laser laser from tissue laser laser laser capture from capture from capture from capture from sections capture from capture from capture from tissue tissue tissue tissue tissue tissue tissue sections sections sections sections sections sections sections aIf you have used more than eight islet preparations, please complete additional forms as necessary bFor example, IIDP, ECIT, Alberta IsletCore cPlease specify the therapy/therapies dTime of islet culture at the isolation centre, during shipment and at the receiving laboratory ePlease specify the test and the results
Version 1.0, created 16 Nov 2018 Page 61 of 63 Diabetes
Checklist for Reporting Human Islet Preparations Used in Research
Adapted from Hart NJ, Powers AC (2018) Progress, challenges, and suggestions for using human islets to understand islet biology and human diabetes. Diabetologia https://doi.org/10.1007/s00125-018-4772-2.
Manuscript DOI: https://doi.org/10.2337/DB19-0979.R1
Title: NKG2D signaling within the pancreatic islets reduces NOD diabetes and increases protective central memory CD8+ T cell numbers
Author list: Andrew P. Trembath, Kelsey L. Krausz, Neekun Sharma, Ivan C. Gerling, Clayton E. Mathews and Mary A. Markiewicz
Corresponding author: Mary A. Markiewicz Email address: [email protected]
Islet preparation 49 50
MANDATORY INFORMATION
Unique identifier 6197 6147
Donor age (years) 22 23.8
Donor sex (M/F) M F
Donor BMI (kg/m2) 28.2 32.9 Diabetes Page 62 of 63
Donor HbA1c or other measure of blood glucose 5.5 5.2 control
Origin/source of isletsb nPOD nPOD
University of University of Islet isolation centre Florida Florida
Donor history of No No diabetes? Yes/No
If Yes, complete the next two lines if this information is available
Diabetes duration (years)
Glucose-lowering therapy at time of deathc
RECOMMENDED INFORMATION
Head Head Donor cause of death Trauma Trauma
Warm ischaemia time (h) Unknown Unknown
Cold ischaemia time (h) Unknown Unknown
Version 1.0, created 16 Nov 2018 Page 63 of 63 Diabetes
Estimated purity (%) N/A N/A
Estimated viability (%) N/A N/A
Total culture time (h)d N/A N/A
Glucose-stimulated insulin secretion or other N/A N/A functional measuremente Handpicked to purity? No No Yes/No Additional notes Islets Islets isolated by isolated by laser capture laser capture from tissue from tissue sections sections
aIf you have used more than eight islet preparations, please complete additional forms as necessary bFor example, IIDP, ECIT, Alberta IsletCore cPlease specify the therapy/therapies dTime of islet culture at the isolation centre, during shipment and at the receiving laboratory ePlease specify the test and the results
Version 1.0, created 16 Nov 2018