Immune cell trafficking to the islets during type 1 diabetes

Summary

Inhibition of immune cell trafficking to the pancreatic islets during type 1 diabetes (T1D) has therapeutic potential, since targeting of T cell and B cell trafficking has been clinically effective in other autoimmune diseases. Trafficking to the islets is characterized by redundancy in adhesion molecule and chemokine usage, which has not enabled effective targeting to date. Additionally, cognate antigen is not consistently required for T cell entry into the islets throughout the progression of disease. However, myeloid cells are required to enable T cell and B cell entry into the islets, and may serve as a convergence point in the pathways controlling this process. In this review we describe current knowledge of the factors that mediate immune cell trafficking to pancreatic islets during T1D progression.

Immune cell migration to the pancreatic islets during type 1 diabetes occurs by transendothelial migration, through a series of steps including rolling, arrest, and crawling on the vascular wall culminating with diapedesis through the endothelial barrier. Migration to the islets is characterized by redundancy in adhesion molecule and chemokine usage, and variable requirement for cognate antigen. However, myeloid cells are required to enable T cell and B cell entry into the islets and may serve as a convergence point in the pathways controlling this process.

Introduction

Type 1 diabetes (T1D) is a T cell‐mediated autoimmune disease that affects more than 19 million people worldwide. It is driven by activated autoreactive T cells within the islets of Langerhans causing the destruction of the insulin‐producing β cells of the pancreas. T1D is characterized by glucose dysregulation and if left untreated is lethal 1. Although insulin supplementation works well to prevent mortality, curative therapies that are effective in the inhibition and reversal of T1D progression are lacking. Furthermore, at the time of diagnosis of T1D, not all the patients’ islets have been destroyed, and maintaining the remaining β cell mass may be critical for preventing complications that occur as a result of T1D. One potential strategy for the treatment of T1D is inhibiting trafficking of immune cells to the islets, as targeting of T cell and B cell trafficking has been effective in other autoimmune diseases such as multiple sclerosis 2. In this review we describe what is known about immune cell trafficking to pancreatic islets during T1D progression.

Progression of T1D within the islets

In order to mediate β cell destruction, T cells must first traffic to the islets and exit the blood stream. Autoreactive T cells initially escape negative selection in the thymus, enter the periphery and traffic to the pancreatic lymph node (PLN) 3. Initiation of T1D is then probably driven by an event of β cell death that activates islet‐resident antigen‐presenting cells (APCs) in a pathogenic manner to cause inflammatory cytokine production and trafficking of β cell antigens to the PLN 3, 4, 5. Under homeostatic conditions in mice there are, on average, seven resident myeloid APCs within each islet 6. While islet antigens can be carried to the PLN by APCs from inflamed islets, it is unclear if islet resident APCs carry islet antigen to the PLN prior to inflammation or if antigens are passively transported via pancreatic lymphatics 5, 6. After T cells become activated in the PLN, islet‐specific T cells recirculate through the lymphatics and the thoracic duct into the bloodstream, and can then infiltrate into the islets by extravasation.

Once T cells infiltrate the islet, additional lymphocytes are constantly being recruited 7. Following establishment of islet inflammation, islet infiltration and destruction is self‐sustaining and does not require the presence of the PLN to prime additional autoreactive T cells 8. The levels of individual islet infiltration have been shown to be highly heterogeneous in both non‐obese diabetic (NOD) mice and T1D patients 9, 10. Within a single pancreas, islets can range from infiltrated to destroyed, and the infiltration state of each islet determines the characteristics of immune cell recruitment to and behavior in that islet 11, 12, 13. We determined that, at early stages of islet infiltration, T cells are restimulated by sustained interactions with CD11c‐expressing APCs, leading to increased interferon (IFN)‐γ production 4, 11, 12. Production of IFN‐γ within the islet creates an inflammatory milieu that causes up‐regulation of vascular adhesion molecules on the islet endothelium and increased chemokine production (see sections below on adhesion molecules and chemokines) 4, 14. These inflammatory changes within the islet allow for increased recruitment of immune cells, including but not limited to T cells, mononuclear phagocytic cells (MNPs) and B cells 4, 15, 16.

MNPs within the islets are made up of dendritic cells (DCs), macrophages and monocytes that have been shown to express the surface marker CD11c within the islets 6. Notably, we showed that CD11c⁺ cells in previously infiltrated islets are required for effective lymphocyte trafficking to the islets 13. It is likely that CD11c⁺ cells within the islets regulate multiple pathways to facilitate extravasation into islets with established infiltration. Once β cells within an islet have been destroyed, immune cells leave the islet and may traffic to other β cell‐containing islets or secondary lymphoid organs.

CD11c⁺ cells serve as gatekeepers for lymphocyte entry into the islets

Our work showed that islet CD11c⁺ cells have a gatekeeper function and are required for the recruitment of both T cells and B cells to infiltrated islets, probably through regulation of multiple chemotactic and inflammatory cues 13. CD11c⁺ cells within the islets produce more than 20 different chemokines that can bind chemokine receptors expressed on islet T cells 13. The precise mechanism by which CD11c⁺ cells facilitate the recruitment of lymphocytes remains unclear; however, during extravasation into the islets, T cells that adhered to the islet vasculature did so in close proximity to vascular‐associated CD11c⁺ cells within the islets 13. It is probable that CD11c⁺ cells are a convergence point for multiple redundant mechanisms to allow for proper immune cell trafficking within infiltrated islets. Perhaps therapeutic targeting to deplete subsets or inhibit the function of islet CD11c⁺ cells may allow for inhibition of lymphocyte trafficking to previously infiltrated islets.

Role of antigen in T cell trafficking to the islets

The role played by antigen in T cell trafficking to the islets has been controversial. This could be due to differences in timing and status of islet inflammation between studies when analyzed. Although still controversial, multiple studies conclude that islet antigen presentation by the islet vascular endothelium is a requirement for initial CD8 T cell trafficking to the islets, but this has not been shown for CD4 T cell trafficking 17, 18. APCs in the islet could also present antigen to intravascular T cells through their ability to periscope into the islet vasculature 15. Initial T cell trafficking to the islets is thought to be dependent on antigen. In NOD.β2M^–/– mice that lack a critical component for major histocompatibility complex (MHC) class I expression, T cell trafficking to the islets and insulitis is significantly delayed 19, 20. However, it is difficult to deduce the role of antigen presentation in trafficking from these studies, as MHC class I expression is also required for CD8 T cell survival and activation.

With respect to CD4 T cell trafficking to the islets, non‐islet antigen hen egg lysozyme (HEL)‐specific 3A9 transgenic T cells do not traffic to the islets unless HEL is expressed in the islets using B10.BR.RIP‐mHEL mice 15. However, it is likely that factors other than antigen are also involved in trafficking to the islets, as antibody blockade of MHC class II only leads to a slight impairment in initial CD4 T cell trafficking to the islets 15. In the B10.BR.RIP‐HEL model, once islets become inflamed changes in expression of proinflammatory cytokines, endothelial vascular adhesion molecules and chemokines allow for non‐islet antigen‐specific T cell trafficking 14. However, an elegant study using NOD retrogenic bone marrow chimeric mice that expressed islet antigen‐specific or non‐specific T cell receptors (TCRs) concluded that antigen is required for long‐term accumulation of T cells within the islets 21, which could result from a combination of trafficking, expansion, retention and survival of T cells.

Once islet infiltration is established, redundant pathways for T cell recruitment allow both islet antigen‐specific and non‐specific T cell trafficking to inflamed islets. To this end, our work clearly demonstrated this to be true by using NOD.C6 mice, which establish islet infiltration and T1D progression equivalent to wild‐type (WT) NOD mice, but lack the antigen for T cells expressing the BDC‐6.9 TCR 22, 23. T cells from NOD.BDC‐6.9 transgenic mice trafficked similarly to the islets of NOD.C6 and WT NOD mice, proving that antigen is not required for T cell trafficking to previously infiltrated islets 13. This is further highlighted by the observation that the majority of T cells that traffic to inflamed islets have a naive phenotype 7, 24. The trafficking of naive cells may be due in part to the presence of tertiary lymphoid organization within inflamed islets in NOD mice 25, 26, 27. Interestingly, non‐islet antigen‐specific T cells within the islets may be suppressive, as these non‐specific T cells lead to decreased activation of islet antigen‐specific T cells and lower disease progression 24. Antigen is not required for the entry of T cells into the inflamed islets, but instead is probably required for the long‐term retention and restimulation of pathogenic T cells within the islets. The requirements for retention of immune cells within inflamed tissues also remain unclear.

Process of lymphocyte extravasation

One of the key regulators of immune cell function is the ability to traffic to sites of inflammation and extravasate from the vasculature into the tissue parenchyma. The process of lymphocyte extravasation into an inflamed non‐lymphoid tissue is a highly regulated process governed by many extracellular and intracellular cues. Prior to trafficking to inflamed non‐lymphoid tissue, lymphocytes are normally activated within the lymph nodes causing up‐regulation of integrins and chemokine receptors that, in many cases, are specific to the sites to which they will home 28, 29, 30. Extravasation of T cells has been studied to a greater extent than that of B cells, but both types of lymphocytes have been shown to use similar mechanisms to traffic to sites of inflammation 31. Extravasation can be broken down into three separate processes: (1) rolling on the vascular wall; (2) firm adhesion and crawling on the endothelium; and finally (3) diapedesis (the process of exiting through the vascular wall) (Fig. 1) 32.

Figure 1.

T cell extravasation into the pancreatic islets. The process of extravasation can be broken down into rolling on the vascular wall, firm adhesion and crawling and diapedesis. T cell extravasation is mediated by molecular interactions between the T cells and the vascular endothelium. (1) Interactions between vascular adhesion molecules, including vascular endothelial (VE)‐cadherin, platelet and endothelial cell adhesion molecule 1 (PECAM‐1) and junctional adhesion molecule (JAM) proteins, maintain tight junctions between endothelial cells forming the vascular barrier. Chemokine‐producing mononuclear phagocytes associate with the islet vasculature. (2) T cells roll on the vascular endothelium by tethering to P‐selectin glycoprotein ligand 1 (PSGL‐1) and L‐selectin through low affinity interactions between T cell‐expressed selectins and cell adhesion molecules on the vasculature. (3) Chemokine signaling causes T cells to arrest on the vascular endothelium by promoting the high‐affinity conformation of integrins such as lymphocyte function‐associated antigen 1 (LFA‐1), very late antigen 4 (VLA‐4) and integrin alpha 4 beta 7 (LPAM‐1), which bind cellular adhesion molecules intercellular adhesion molecule 1 (ICAM‐1), vascular cell adhesion molecule 1 (VCAM‐1) and mucosal vascular addressin cell adhesion molecule 1 (MadCAM‐1), respectively. (4) T cells crawl on the vasculature following chemokine gradients to find a permissive site of extravasation in proximity to mononuclear phagocytes. (5) Integrins on T cells interact with endothelial junction proteins to disrupt the vascular barrier and facilitate diapedesis. Chemokine and integrin signaling also lead to rearrangement of the actin cytoskeleton, which allows the T cell to squeeze through the endothelial junctions. (6) T cells complete extravasation to enter the islet parenchyma and the endothelial barrier is restored.

Our laboratory recently characterized the process of T cell extravasation into the islets using intravital 2‐photon imaging 13. We found that the majority of T cells that adhere to the islet vasculature failed to complete the process of diapedesis within our 2‐h imaging period, with a subset releasing back into the bloodstream. T cells that completed diapedesis into the islet parenchyma took more than an hour to complete the process. Furthermore, within our 2‐h imaging window, completion of extravasation was only observed in islets with advanced levels of T cell infiltration, suggesting that the process of extravasation takes longer than 2 h in islets that are at early stages of infiltration 13. As a point of comparison, timing of T cell extravasation into the islets is more similar to extravasation into the highly restrictive central nervous system than into the permissive sites such as the lymph nodes, which takes only 5–10 min 33, 34. These data suggest that the islet vasculature is a highly restrictive site for T cell entry.

In the vasculature at the site of inflammation, T cells are captured by vascular adhesion molecules and roll on the vascular lumen. This tethering and rolling is caused by interactions of P‐selectin glycoprotein ligand 1 (PSGL1) or L‐selectin on lymphocytes binding P‐ and E‐selectins or mucosal vascular addressin cell adhesion molecule 1 (MAdCAM1), respectively, on the inflamed endothelium while under shear force 32 (Fig. 1). Chemokine receptors on activated T cells then bind chemokines bound to the inflamed vascular endothelium. Chemokine receptor signaling in activated T cells causes up‐regulation of integrin affinity through a process referred to as ‘inside‐out signaling’ 35, 36. High‐affinity integrins on activated T cells bind to the endothelial expressed cellular adhesion molecules (CAMs) 32. The high‐affinity integrin interaction with their ligands causes T cells to arrest on the vascular endothelium (Fig. 1). T cells then crawl on the vascular endothelium following chemokine gradients to find a permissive site of entry before they undergo diapedesis 32, 36. T cell diapedesis involves integrin‐ and chemokine‐driven cytoskeletal rearrangements within the T cells, loosening of the endothelial junctions of the vascular wall and breakdown of the endothelial basement membrane to successfully extravasate into the inflamed tissues 32, 37 (Fig. 1). These processes can vary greatly, depending on the site of T cell priming, T cell subset polarization, endothelial adhesion molecule expression and the site of inflammation. Although there is a high potential for therapeutic intervention by inhibiting T cell entry into inflamed islets, the mechanisms of T cell trafficking and extravasation into the islets during T1D are still not well understood.

Role of vascular adhesion molecules in T cell trafficking to the islets

Vascular adhesion molecules are involved in leukocyte adhesion to the vasculature within sites of inflammation, which is necessary for extravasation 32. The initial step of tethering, or leukocyte capture by the vascular endothelium, during leukocyte extravasation is mediated by lower‐affinity interactions of selectins with their ligands causing rolling on the vascular endothelium 38. These interactions include: (1) L‐selectin (CD62L) on leukocytes binding to the mucosal adhesion molecule MAdCAM or to the high endothelial venule (HEV) adhesion molecules glycosylation‐dependent cell adhesion molecule‐1 (GlyCAM) and peripheral node addressin (PNAd) on the vasculature; (2) PSGL‐1 on leukocytes binding vascular P‐selectin, L‐selectin or E‐selectin; and (3) homotypic binding of E‐selectin on both lymphocytes and the vasculature 30, 32 (Fig. 1, Table 1).

Table 1.

T cell adhesion molecules in T1D

Receptor	Ligands	Ligand expression in islets		Role in T1D
		Animal models	Human
L‐selectin	MAdCAM1	Low–high 41, 44	Low–high 42, 43	NOD mice lacking L‐selectin progress to T1D 41 L‐selectin high T cells in the islets of NOD mice have regulatory function 44 T1D patients have decreased levels of L‐selectin on memory T cells and increased serum levels of cleaved sL‐selectin 42, 43
	GlyCAM
	PNAd
PSGL‐1	Selectins	High 45	?	Blockade of PSGL‐1 leads to reduced T1D in NOD mice through increased T cell apoptosis 45
LFA‐1	ICAM‐1	Mid 14, 48, 50, 108	+ 42, 46, 100	LFA‐1 deficiency or blockade inhibits T1D progression in NOD mice 47, 48, 49 ICAM‐1 deficiency prevents T1D in NOD mice, and antibody blockade reduces progression of T1D 14, 49, 50
VLA‐4	VCAM‐1	High 14, 48	+ 42, 100	Early blockade of α4 integrins and VCAM1 reduced T1D progression in NOD mice 48
LPAM‐1	MAdCAM‐1	High 39, 51, 52	+ 42, 100	Blockade of MAdCAM prior to islet infiltration reduced T1D progression, but had no effect once islet infiltration was established in NOD mice 51, 52

T1D = type 1 diabetes; MAdCAM‐1 = mucosal vascular addressin cell adhesion molecule 1; GlyCAM = glycosylation‐dependent cell adhesion molecule‐1; PNAd = peripheral node addressin; PSGL‐1 = P‐selectin glycoprotein ligand 1; LFA‐1 = lymphocyte function‐associated antigen 1; VLA‐4 = very late antigen 4; LPAM‐1 = integrin alpha 4 beta 7; NOD = non‐obese diabetic.

Although islet‐infiltrating lymphocytes express a variety of selectins and the islet vasculature expresses their corresponding ligands, none of these initial tethering molecules are required for T cell homing to the islets during T1D 4, 39. Combinatorial blockade of L‐ and P‐selectin does not inhibit T1D in transfer models, and there is no literature implicating E‐selectin in lymphocyte trafficking to inflamed islets 40. Furthermore, NOD mice deficient in L‐selectin progress normally to T1D 41. In patients with T1D, serum levels of L‐selectin are elevated and memory T cells have decreased L‐selectin, suggesting increased activation of memory T cells during T1D 42, 43. Interestingly, CD4 T cells that express high levels of L‐selectin and traffic to the islets have been shown to have regulatory function within the islets 44. Blockade of PSGL‐1 inhibits T1D progression but not through blockade of islet trafficking. Instead, crosslinking of PSGL‐1 was shown to cause lymphocyte apoptosis 45. Although these interactions are probably involved in lymphocyte trafficking to the islets, it seems that there must be alternative or redundant pathways that allow for lymphocyte tethering and rolling in the islets.

The second step of extravasation is integrin‐mediated adhesion to the vasculature. Chemokine signaling in T cells rolling on the vasculature drives increased integrin affinity leading to integrin ligation, T cell arrest and crawling on the vasculature. Within the serum of T1D patients and islets of NOD mice, T cells have been shown to express the integrins lymphocyte function‐associated antigen 1 (LFA‐1) (αLβ2), very late antigen 4 (VLA‐4) (α4β1) and integrin alpha 4 beta 7 (LPAM‐1) (α4β7), and the islet vasculature expresses their respective ligands intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1) and MAdCAM1 4, 39. Cytokines produced locally in the islets or present systemically in the serum of T1D patients and NOD mice concurrently drive the expression of the integrin ligands, ICAM1, VCAM1 and MAdCAM1 in the islet vasculature 4, 14, 15, 46. Although multiple integrins seem to play a necessary role in initial T cell trafficking, once infiltration is established even these adhesion molecules can sometimes become redundant. NOD mice that are deficient in αL or β2, the two chains of LFA‐1, or treated with antibody blockade of α4 integrins prior to islet infiltration do not progress to T1D 47, 48, 49. Notably, in the context of T1D, the role of αL and β2 integrins appear different, with αL deficiency probably having a dominant effect on T cell activation and β2 deficiency directly affecting adhesion to islet vasculature 47. These differences may be due to the ability of β2 integrin to form dimers with other α chains to form additional integrin pairs. ICAM‐1, the ligand for LFA‐1, also has a dominant role early in T1D progression, as NOD mice deficient in ICAM‐1 do not develop T1D, but this may be caused by an impairment of T cell activation 50. Once islet infiltration is established, blockade of α4 integrins or ICAM‐1 strongly reduces T1D progression, but approximately 20–40% of mice still progress to T1D 14, 49. Antibody blockade of MAdCAM‐1 prior to islet infiltration also strongly reduces T1D disease incidence, although some mice were still able to progress to T1D, and once islet infiltration occurred it no longer inhibited disease progression 51, 52. While these studies are important in understanding the requirements for T cell trafficking to the islets, blockade of all of these selectins and integrins would be impractical and probably lead to global immunosuppression.

Role of chemokines in leukocyte trafficking to the islets

Chemokines are cytokines that are responsible for directing migration, extravasation and positioning of immune cells throughout the body. The chemokine superfamily is made up of 46 members in humans, many of which have homologs in mice 53. Chemokines are named and grouped together based on the position of conserved cysteine residues into CC and CXC ligands and receptors, with a few outliers such as XC and CX3C motifs 54. Between mice and humans, more than half the chemokine ligands and receptors have been implicated to have a role in T1D 55 (Table 2). Many of the chemokines produced within the islets are IFN‐stimulated genes driven by IFN‐γ 4, 14, 55, 56, 57, 58, 59, 60. Our laboratory and others have shown that chemokine receptor signaling on T cells is necessary for T cell recruitment to previously infiltrated islets 13, 15. Furthermore, the role of chemokines in the progression of T1D was elegantly highlighted by expressing the gammaherpesvirus‐68 chemokine decoy receptor, M3, within the islets. The M3 protein broadly blocks binding to multiple CC chemokines inhibiting their chemotactic function 61, 62. NOD.M3 mice exhibited inhibited immune trafficking to the islets and did not progress to T1D 63. Unfortunately, while targeting of individual chemokine pathways has been effective at delaying trafficking to uninfiltrated islets, it does not seem to be effective in inhibiting trafficking once infiltration is established 13, 57, 64, 65, 66. This is due probably to the fact that chemokines are highly redundant and promiscuous, being able to bind multiple chemokine receptors on a variety of cell types 4, 14, 55, 56, 57, 58, 59, 60.

Table 2.

Roles of islet‐expressed chemokines in type 1 diabetes

Receptor	Ligands	Ligand expression in islets		Role in T1D
		Animal models	Human
CCR2	CCL2	Mid 4	Mid 60, 102, 109	NOD and C57Bl/6 T1D models show contradictory outcomes CCR2–/– NOD mice have impaired T1D progression 69 Transgenic expression of CCL2 in the islets of C57Bl/6 mice induced T1D penetrance 70 Transgenic expression of CCL2 in the islets of NOD mice reduces T1D penetrance 71
CCR5	CCL3,	Mid 63	Low/neg 60	CCR5–/– NOD mice have accelerated T1D 69 CCL3–/– NOD mice have impaired T1D progression 72
	CCL4,	Mid 63	Low/neg 60
	CCL5	Mid 55	Mid 55
CCR7	CCL19,	Mid 110		CCR7–/– NOD mice do not progress to T1D 79
	CCL21	Mid 110
CXCR3	CXCL9,	Mid 13, 55, 57	Mid 55, 60	NOD and C57Bl/6 T1D models show contradictory outcomes CXCL10 neutralizing antibody can delay and reverse T1D in NOD mice 65, 83 Transgenic CXCL10 expression in C57Bl/6 islets can drive islet infiltration 84 Virally induced T1D in C57Bl/6 is impaired when T cells lack CXCR3 82 CXCR3–/– NOD mice get accelerated T1D 66 CXCR3 deficient T cells are able traffic effectively to infiltrated islets in NOD mice 13
	CXCL10	High 13, 55, 57, 64, 82	High 55, 60
CXCR4	CXCL12	Mid 89, 90, 91	Mid 60	Blockade of CXCL12 in short‐term disease transfer leads to accelerated disease 89. Long‐term blockade of CXCL12 reduces T1D progression in NOD mice 90
CXCR5	CXCL13	Mid 25, 111		Transgenic expression of CXCL13 in islets drives islet infiltration in C57Bl/6 mice 97 Antibody blockade of CXCL13 disrupts tertiary lymphoid organization in the islets in NOD mice 25
CXCR6	CXCL16	High 13		CXCL16 is highly expressed in islet myeloid cells in NOD mice 13 CXCR6 deficient T cells traffic to islets normally in NOD and C57Bl/6 13 CXCR6 is located within the IDDM22 T1D risk locus in human 94, 95 CXCL16 is a candidate gene for the Idd4 T1D risk locus in mouse 93
M3 viral decoy receptor	CC chemokine ligands			Islet expression of M3 blocks T1D progression in NOD mice 63

CCR = chemokine receptor; CCL = CC chemokine ligand; NOD = non‐obese diabetic; T1D = type 1 diabetes; CXCR = C‐X‐C chemokine receptor; CXCL = C‐X‐C motif) ligand 1; Idd = insulin‐dependent diabetes.

Chemokines in MNP trafficking to the islets

Chemokine‐driven recruitment of MNPs to the islets has a complex role in T1D progression. MNPs within the islets are highly heterogeneous, with both pathogenic and tolerogenic populations 6, 11, 12, 67, 68. Two of the major chemokine receptors that are responsible for MNP recruitment are chemokine receptor (CCR)2 and CCR5. CCR2 binds the chemokine CC chemokine ligand (CCL)2, whereas CCR5 ligands include CCL3, CCL4 and CCL5 53. Interestingly, deficiencies in CCR2 or CCR5 in NOD mice have countervailing effects during T1D progression. CCR2 deficiency inhibits T1D progression, whereas CCR5 deficiency leads to increased MNP recruitment and accelerated T1D progression 69. This regulation becomes even more complicated when also considering the role of the chemokine ligands, as well as mouse strain susceptibility to T1D. CCL2 expression within the islets of diabetes‐resistant mice (C57BL/6 × DBA.RIP‐CCL2) caused the recruitment of MNPs, insulitis and T1D progression, and these effects were lost with CCR2 deficiency 70. Conversely, CCL2 expression in the islets of NOD mice (NOD.RIP‐CCL2) resulted in recruitment of tolerogenic MNPs leading to lowered insulitis and inhibited T1D progression 71. Furthermore, the deletion of CCL3 in NOD mice led to decreased insulitis and delayed T1D, which contradicts the data on the deletion of its receptor CCR5 69, 72. These studies highlight the complexity of the MNP populations and their recruitment to the islets. Notably, as MNP populations in the islets are required for T cell entry into the islets 13, recruitment of the islet MNP populations may, in turn, affect T cell recruitment to the islets.

Chemokines in T cell trafficking to the islets

The chemokine receptor CCR7 and its ligands CCL19 and CCL21 are known to be important for immune cell recruitment to, and localization within, the lymph nodes 73, 74, 75, 76. However, during T1D in NOD mice, CCL19 and CCL21 are also expressed around prediabetic islets 77. This could be due in part to the presence of tertiary lymphoid organs (TLOs) within the islets. This expression of CCL21 around the islets has been shown to be important for homing of islet antigen‐specific T cells to the islets 17. The receptor CCR7 has also been associated with the insulin‐dependent diabetes (Idd)9 T1D risk allele in NOD mice, which is involved with homing of T cells to the islets 78. Furthermore, NOD mice deficient in CCR7 do not develop T1D 79, highlighting its role in the initiation of islet‐specific disease probably through its function in both the lymph nodes and the islets.

Another major chemokine receptor‐ligand pathway that has been well studied in the progression of T1D is C‐X‐C chemokine receptor (CXCR)3 binding to C‐X‐C motif ligand (CXCL)9 and CXCL10 produced within the islets. CXCR3 also binds CXCL11, which is not functional in C57Bl/6 mice, but may be carried forward in backcrosses from the 129 background, potentially complicating interpretations of chemokine knockout analyses 80. CXCR3 is expressed on T helper type 1 (Th1) CD4 and effector CD8 T cells, and expression of its ligands is driven by IFN‐γ 81. The receptor CXCR3 is important for T cell trafficking to a variety of sites of anti‐viral and autoimmune inflammation 81. CXCL9 is produced within the islets of NOD mice, and β cells have been shown to be the major producer of CXCL10 in the islets 57, 82. Neutralization of CXCL10 in NOD mice can delay and sometimes reverse T1D progression 65, 83. Furthermore, expression of CXCL10 in the beta cells of T1D‐resistant mice can drive insulitis and cause accelerated virally induced T1D 84. Also, during virally induced T1D, mice deficient in CXCR3 had reduced disease onset 82. Conversely, NOD mice deficient in CXCR3 exhibited an accelerated rate of T1D, due probably to the inhibition of regulatory T cell trafficking to the islets 66, 85, 86. Our work has shown that T cell‐specific deficiency of CXCR3 is not sufficient to prevent T cell trafficking to inflamed islets in the RIP‐mOva model 13. CXCR3 and its ligands may be involved in early T cell trafficking to the islets as well as trafficking of T_regs, but are not necessary for effector T cell trafficking and T1D progression.

Another chemokine that has both tolerogenic and pathogenic roles in T1D pathogenesis is CXCL12 and its receptor CXCR4. The receptor CXCR4 is highly expressed on most immune cells and has an important role in the maintenance of hematopoietic stem cells within the bone marrow 87, 88. In T1D, CXCR4⁺ T cells have been shown to be regulatory and can inhibit disease transfer into NOD mice. In these disease transfer models, blockade of CXCL12 led to increased insulitis and accelerated T1D progression 89. CXCL12 has been shown to be chemorepulsive to diabetogenic T cells in vitro by blocking up‐regulation of high affinity integrins and strong binding to the islet endothelium 90. However, CXCL12 expression in the islets of C57BL/6 mice protects β cell survival after streptozotocin treatment 91. Additionally, long‐term blockade of CXCL12 in NOD mice reduced T1D disease progression 92. The differences in these results are likely be due to differential recruitment of effector and regulatory T cells to the islets at different stages of T1D progression.

There are many other chemokines that probably play redundant roles in the recruitment of T cells to the islets, including CXCL16 and its receptor CXCR6. This chemokine ligand‐receptor pair is of particular interest in T1D, as CXCL16 is a potential candidate gene for the Idd4 T1D risk locus in mice, and CXCR6 is located within the IDDM22 T1D risk locus in humans 93, 94, 95, 96. In our work, CXCR6 was the highest chemokine receptor mRNA transcript expressed in islet T cells, and CXCL16 was the third highest expressed chemokine transcript in islet CD11c⁺ cells 13. CXCL16 protein is selectively expressed in the islets by CD11c⁺ cells 13. Although this chemokine ligand‐receptor pair probably contributes to recruitment of T cells by islet CD11c⁺ cells, NOD.CXCR6^–/– mice progressed to T1D similarly to WT NOD mice 13. NOD.CXCR6^–/– T cells also trafficked normally to the islets of WT NOD mice with established islet infiltration. Surprisingly, even C57Bl/6.CXCR3^–/–CXCR6^–/– T cells trafficked normally to the islets of C57Bl/6.RIP‐mOVA mice with established islet infiltration 13. These data highlight the high level of redundancy in chemokines that are able to promote T cell trafficking to the islets once islet inflammation is established.

Chemokines in B cell trafficking to the islets

B cell recruitment to the islets during T1D is also probably driven by chemokines. The chemokine CXCL13 binds the receptor CXCR5, which is highly expressed on most B cells 53. Expression of CXCL13 in diabetes‐resistant mice causes insulitis and B cell‐driven TLO formation within the islets 97. Antibody blockade of CXCL13 in NOD mice disrupts TLO formation in the islets but does not affect T1D disease progression 25. This shows both that B cells can be recruited to the islets by chemokine expression and that one of the roles that B cells play during T1D progression is maintenance of TLOs in the islets.

Biomarkers of islet trafficking in T1D

Understanding biomarkers of T1D progression has also been of great scientific interest. Molecules involved in leukocyte trafficking, including chemokines and elevated serum levels of soluble adhesion molecules, may represent promising biomarkers to understand immune cell activation and progression of islet infiltration during T1D 42, 43, 98, 99, 100, 101. Patients with T1D have been shown to have elevated serum levels of inflammatory chemokines, including CCL2 and CXCL10 98, 102, 103, 104. Upon early onset of T1D, there is also a reduction in peripheral blood leukocytes expressing Th1 chemokine receptors, such as CCR5 and CXCR3 105. This reduction is thought to be due to the recruitment of peripheral lymphocytes to the islets during disease onset. Decreased levels of L‐selectin on memory T cells and increased serum levels of cleaved sL‐selectin in T1D patient serum could be biomarkers of increased T cell activation 42, 43. Serum chemokine levels may be useful in understanding the subtle changes of the immune response during clinical trials in conjunction with other accepted biomarkers for disease progression. Some of these readouts could potentially be added to established prognostic biomarkers for disease progression and response to interventions such as serum c‐peptide levels, islet autoantibody expression, T cell phenotype, HbA1c, and serum blood sugar 98, 101.

Concluding remarks

The only current treatment for T1D is insulin replacement. While insulin replacement is effective in treating T1D symptoms, it does not cure the underlying autoimmunity that drives the disease. Inhibition of immune cell trafficking to diabetic islets has the potential to intervene in the underlying immune dysfunction that leads to T1D, but this strategy has not yet been effective.

Multiple redundant pathways, particularly with relation to chemokines, are involved in the trafficking of immune cells to diabetic islets, as well as in normal immune cell homeostasis and function during inflammation and infection. Many chemokines and chemokine receptors are located within T1D risk alleles 56, 93, 106. Several chemokines that have been shown to be elevated during T1D progression have not yet been well studied 14, 15, 55. Also, some of these chemokines, such as CCL22, are thought to be more involved in trafficking to the PLN than in trafficking to the islets 107. Other chemokines still have unknown or redundant functions. Despite the therapeutic potential of targeting chemokines and their receptors, the high level of redundancy, as well as the important role of chemokines in normal immune function, make this a challenging avenue of research.

Effectively targeting these redundant islet homing pathways while avoiding the induction of broad immunosuppression caused by non‐specific inhibition of lymphocyte trafficking is a major unaddressed challenge. Our work identifying CD11c⁺ cells as gatekeepers for lymphocyte trafficking to the islets may provide a convergence point for targeting multiple pathways utilized for lymphocyte entry into the islets, but would require further research to enable specific targeting of pathogenic CD11c⁺ populations without driving systemic immunosuppression. While of great interest, much work must still be conducted in order to understand the mechanisms that are required for immune cell trafficking to the islets during T1D.