T1D is a tissue specific and T cell–mediated autoimmune disease characterized by the inflammation of pancreatic islets, namely insulitis, resulting in selective destruction of insulin-producing beta islet cells and development of overt diabetes (Atkinson & Leiter, 1999, Bach, 1994, Mathis et al., 2001, McDevitt, 2001, Nepom & Kwok, 1998, Quinn et al., 2001). The onset of T1D is preceded by at least two main inflammatory stages during insulitis development (Bach, 1994, Robles et al., 2003). In the first stage, termed peri-insulitis, a mixed population of leukocytes, including macrophages, dendritic cells, and T cells, migrate from draining lymph nodes to the peripheral space to the islets. In the second stage, the leukocytes further migrate and infiltrate into the islets, resulting in invasive insulitis. Overt T1D will develop when the majority of insulin-producing β cells in the islets are destroyed by the invading leukocytes and insufficient insulin is produced to control blood glucose levels in the body. Among leukocytes that infiltrate the islets, the autoantigen-specific diabetogenic T cells play a critical role in development of T1D. Recruitment or homing of these diabetogenic T cells into the islets is a critical component of insulitis leading to T1D. Thus inhibition of their homing to the islets would prevent the development of insulitis and T1D. Consequently, it is necessary to understand the homing or trafficking behavior of diabetogenic T cells during the formation of peri-insulitis and invasive insulitis. The knowledge gained from studies addressing those questions is imperative to the development of early diagnosis methods and immune modulatory approaches to treat T1D.
Due to the low number and frequency of T cells specific for an autoantigenic peptide, it has been difficult to identify and trace autoantigen-specific T cells in animals. We have previously addressed this problem by generating novel MHC class II tetramers that can stain T cells specific for a self peptide recognized presented by the MHC class II molecules such as the I-Ag7 in NOD mice (Liu et al., 2000, Chen et al., 2003, You et al., 2003). Using this approach, we have been able to successfully identify and trace autoantigen-specific T cells, such as the CD4+ BDC2.5 (BDC) T cells. The tissue localization of various T cell populations at any given time point can be identified using techniques such as flow cytometry and immunofluorescence. However, these methods involve an invasive approach requiring tissue removal from animals, and therefore only provide terminal data. As such, these methods cannot help us assess real-time dynamic cell migration patterns
Previous studies have shown that it is possible to monitor T cells and other immune cells in deep organs in small animals (Weissleder, 1999). In particular, positron-emission tomography imaging (PET) of radio-labeled cells (Oku et al., 1994, Koike et al., 1997, Kikkawa et al., 2000, Melder et al., 2002, Adonai et al., 2002) and magnetic resonance imaging (MRI) of probe-labeled cells (Lewin et al., 2000, Dodd et al., 2001, Moore et al., 2002) have been used to image cell trafficking in small animals. In addition,
Over the past decade, new imaging technologies for monitoring molecular and cellular changes in living animals have been developed. These methods were developed to help reveal the
In the following sections, we will review some of the results obtained from
In vivoimaging of pathogenic T cell-mediated insulitis during development of overt type 1 diabetes
Despite extensive studies on the role of T cells in T1D, their
2.1. Real time bioluminescent imaging analyses
Previous studies have shown that BLI analyses can be used to view cell trafficking in small animals, using luciferase (Luc) as an
2.2. Noninvasive real-time
in vivobioluminescence imaging of diabetogenic T cell trafficking patterns
The CD4+ BDC2.5 (BDC) cells are islet antigen–specific and highly diabetogenic T cells (Haskins and McDuffie, 1990, Haskins et al., 1989). Activated BDC cells from BDC TCR transgenic NOD mice (BDC mice) induce an aggressive form of diabetes (Chen et al., 2006, Katz et al., 1995). To label T cells from these animals for trafficking studies, we crossed a transgenic reporter mouse line that expresses luciferase by a strong, ubiquitous, constitutive promoter with BDC mice for more than 8 generations (luc-BDC/NOD mice) (Cao et al., 2004). This synthetic hybrid promoter is composed of the chicken β-actin promoter and the immediate early enhancer from cytomegalovirus, and thus drives expression of Luc into virtually all cells in the mice (Cao et al., 2004). Therefore, transgenic expression of the Luc transgene in BDC mice results in stable expression of Luc in all BDC cells. Using these Luc-expressing BDC/NOD mice allows for long-term longitudinal
It is known that transferring CD4+ BDC cells alone into NOD/scid mice failed to induce diabetes. However, cotransfer of CD4+ cell–depleted splenocytes isolated from diabetic NOD mice with CD4+ BDC cells induced accelerated diabetes in recipient mice. It has also been observed that there can be a respectful degree of peri-insulitis or invasive insulitis (Katz et al., 1995, Katz et al., 1993, Peterson & Haskins, 1996). However, the mechanisms responsible for these novel observations were not clear. At least two non-mutually exclusive possibilities exist that may explain the effect of the non-CD4 splenocytes on CD4+ BDC cells during induction of expedited diabetes: (1) The non-CD4 splenocytes may cause an altered systemic trafficking pattern of BDC cells that prevents their homing to the islets, or (2) they may exert their effect on BDC cells locally at tissue levels. We have addressed these questions in our studies which will be discussed in more detail below.
We imaged BDC cells
Using an adoptive transfer model, we have imaged Luc+ CD4+ BDC cells and examined their trafficking patterns in real time in NOD/scid mice recipients (Fig. 1). In order to monitor diabetogenic T cell trafficking patterns in real-time
To quantify the bioluminescent signals detected from various tissues in the recipient mice, the total flux (photons/sec) was determined by drawing regions of interest (ROI) over the body corresponding to the designated organs. Based on these results, we have determined the total photon output per ROI to calculate the bioluminescence signal intensity. Each ROI was visually defined to be specific areas that correspond to selected organs from both recipient animals and control mice. The rates of increase in the
In summary, these noninvasive
2.3. Real time bioluminescent imaging of diabetogenic T cells during the development of insulitis leading to diabetes
The fact that a large number of Luc+ CD4+ BDC cells present in the islets did not induce diabetes in the recipient mice was not due to the introduction of luciferase transgene into BDC mice. This is because purified Luc- CD4+ BDC cells obtained from BDC mice of the same age in our mouse colony also did not induce diabetes in recipient mice (unpublished data). One possible explanation of these results is that the BLI analyses study was terminated before overt diabetes developed; however, the control mice receiving whole BDC mouse splenocytes develop diabetes within 6 weeks after cell transfer (Chen et al., 2006). This explanation seems unlikely because our additional studies have shown that recipient mice did not develop diabetes even at 23 weeks following the transfer of BDC cells alone (unpublished data). Alternatively, it is possible that BDC cells require the presence of other types of cells in the spleen in order to destroy the islets and induce diabetes.
It has been shown that transfer of T cells alone did not induce diabetes, and the presence of non-CD4+ cells such as CD8+ T cells are necessary (Bendelac et al., 1987, Christianson et al., 1993, Miller et al., 1988). Previous studies have also shown that cotransfer of the CD4+ BDC cells with CD4+ cell–depleted splenocytes from newly diabetic NOD mice induced accelerated diabetes in the recipient animals (Peterson and Haskins, 1996). In order to understand the trafficking behavior of BDC cells during islet inflammation that leads to diabetes, we co-transferred purified CD4+ Luc+ BDC cells and CD4+ cell–depleted Luc- splenocytes from newly diabetic NOD mice into NOD/scid recipient mice. Our results showed that the recipient mice receiving non-CD4+ splenocytes alone did not develop diabetes. However, the mice that received both populations of cells developed diabetes as early as 9 days following cell transfer, and all recipient mice developed accelerated diabetes within 3 weeks.
In order to understand the behavior of BDC cells in these mice, BLI studies showed that the kinetics of Luc+ BDC cells’ trafficking in mice that were cotransferred with both CD4+ Luc+ BDC cells and CD4+ cell–depleted Luc- splenocytes from newly diabetic NOD mice were similar, at the level of the whole body and organs, to the kinetics in recipient mice that have BDC cells alone. In both types of recipients, the BDC cells migrated first to the lung in one day and then to the pancreas, inguinal lymph nodes, and spleen within one week. To further quantify and compare the changes in BLI signal intensity (total flux) in various tissues of the two different groups of recipient mice at the same time points, we imaged BDC cells and analyzed the ROI drawn on various tissues including the pancreas. The ROI analyses showed that the total BLI signal intensity gradually increased over time. A similar trend of signal intensity increase was also detected in the pancreas ROI in both cohorts, although the transferred Luc+ BDC cells continued proliferating in the lymph organs in mice that received both cell types. Comparable levels of BLI intensity over time were detected in the lymphoid organs in both groups of mice before all the mice that received both types of cells became diabetic following a cotransfer of the cells. However, the BLI intensity in lymphoid organs increased at a faster rate in co-transferred mice during the post-diabetic period than that detected in nondiabetic mice that received BDC cells alone. Therefore, cotransfer of CD4- splenocytes led to diabetes but did not otherwise change the overall trafficking patterns of BDC cells to these tissues during the pre-diabetic stage. These results suggest that the co-transferred splenocytes may not help BDC cells to induce accelerated diabetes by modulating the trafficking and the total number of BDC cells in the pancreas. Instead, they may cause an increased proliferation of BDC cells in tissues other than the pancreas. BDC cells were able to induce insulitis in both groups of recipients, but only the co-transferred recipients developed diabetes. Therefore, it is likely that the total number of BDC cells present in the pancreas is not responsible for the development of diabetes. It may be that the cellular function of immune cells present within the islets and the microscopic localization of the cells within the tissue were the contributors to diabetes development in our studies.
Different degrees and types of insulitis may occur in the recipient animals after transfer of varied cell populations. Peri-insulitis does not lead to islet destruction, whereas invasive insulitis destroys islets and leads to diabetes. To further evaluate why the cotransferred CD4- splenocytes may help BDC cells induce accelerated diabetes, we examined whether these splenocytes promote infiltration of BDC cells into the islets and induce invasive insulitis. To make these distinctions, we performed microscopic imaging of the islets (since there is no macroscopic imaging method able to distinguish cell distribution differences at this resolution). Initial ex vivo BLI analyses of excised pancreases showed that comparable bioluminescent signals were detected in the pancreas of both groups of recipients (Lee et al., 2007). These results demonstrated that BDC cells can traffic to the pancreas independent on the presence of other splenocytes. Additonal histological analyses showed that mice co-transferred with both cell types developed more severe invasive insulitis than mice that received BDC cells alone. The islets in the pancreatic sections obtained from co-transferred mice on day 20 following cell transfer showed invasive insulitis and were severely damaged. Only peri-insulitis were detected in the islets from mice transferred with BDC cells alone and their islets remained intact. Therefore, the co-transferred splenocytes may help induce diabetes by enhancing the local infiltration of BDC cells into the islets.
We also performed FACS analyses to better understand the cellular component of the invasive insulitis found in the co-transferred mice. We found that the percentage of both CD11b+ cells and CD11c+ cells showed increase only in the islets of co-transferred mice (Lee et al., 2007). These results suggest that these innate immune cells are involved in promoting invasiveness of BDC cells into the islets. One possibility is that these cells help activation of BDC cells, leading to more activated BDC cells in the islets of co-transferred mice and resulting in accelerated diabetes. Investigation on the activation status of BDC cell present in the islets show that there was no difference in expression of activation markers, CD69 and CD62L, on BDC cells isolated from the islets of both cohorts. Overall, our results suggest that efficient trafficking of BDC cells alone to the pancreas is unable to induce T1D alone. The presence of CD4- splenocytes is necessary to help these pathogenic T cells locally infiltrate the islets without altering their activation status and systemic trafficking patterns.
In summary, these findings are possible because investigators have the ability to image T cell trafficking and tissue localization over a range of varied scales of techniques, from macroscopic imaging and whole-body BLI, to microscopic imaging via light microscopy of tissue sections and single cell analyses using FACS.
2.4. Application of BLI to understand the role of nTreg cells in regulating the
in vivobehavior of diabetogenic T cells during T1D
The naturally-arised Foxp3+ CD4+ CD25+ nTreg cells play a critical role in immune tolerance induction and autoimmune disease prevention (Sakaguchi and Powrie, 2007). It is well known that a deficiency in nTreg cells may contribute to development of insulitis and T1D in both humans and mice. Rebuilding a sufficient number of nTreg cells can re-induce immune tolerance, leading to inhibition of T1D (Herman et al., 2004, Tang et al., 2004, Tarbell et al., 2004, Brusko et al., 2005, Lindley et al., 2005, Tritt et al., 2008). Extensive studies have been performed to understand the role of nTreg cells during T1D. However, it is still largely unclear as to how these potent nTreg cells prevent destruction of the insulin-producing beta islet cells by pathogenic T cell-mediated immune responses.
As discussed in previous sections, the development of insulitis precedes development of overt T1D, and immune tolerance induced by nTreg cells often leads to prevention of insulitis in diabetes-free mice. It is conceivable that the development of insulitis involves at least four stages as outlined and depicted in Figure. 2. These include: (1) migration of antigen-presenting cells (APCs), such as macrophages and dendritic cells, to islet draining lymph nodes where the APCs can pick up islet antigens and present them to T cells; this leads to activation of autoreactive pathogenic T cells such as BDC cells in the lymph nodes, (2) expansion of activated pathogenic T cells, (3) migration of activated pathogenic T cells to the islets, and (4) infiltration of the pathogenic T cells into the islets, causing invasive insulitis and destruction of the islets. It is possible that in order to inhibit insulitis and T1D, nTreg cells may function by blocking pathogenic T cells at one of these four stages. For example, nTreg cells may modulate systemic trafficking behavior of pathogenic T effector cells such as BDC cells as well as APCs during the pre-diabetic stage. In addition to suppressing pathogenic T effector cell proliferation and expansion, nTreg cells may block
the trafficking and homing of such T cells to the islets, resulting in inhibition of invasive insulitis that causes overt T1D. It is also possible that, in the absence of nTreg cells, the pathogenic T cells such as BDC cells may traffic more rapidly to and accumulate faster in the islets during pre-diabetic stage. This eventually leads to destruction of the insulin-producing islet beta cells and onset of diabetes.
In our studies to test these different hypotheses, we have performed several experiments, including real time imaging studies, to investigate whether BDC cells showed an altered trafficking pattern in nTreg cell-deficient mice compared to that in nTreg cell-sufficient mice. In order to examine the role of nTreg cells
As part of our studies to address these questions, we used a cell adoptive transfer model by transferring CD4+ BDC cells into NOD/scid recipient mice. It is known that the transfer of CD4+ CD25- (nTreg-deficient) BDC cells into NOD/scid mice induces aggressive T1D, whereas the transfer of CD4+ (nTreg cell-sufficient) BDC cells does not cause onset of T1D in recipient mice. Therefore, to longitudinally monitor trafficking of the adoptively transferred BDC cells in mice with or without nTreg cells, we have isolated Luc+ CD4+ CD25- (nTreg-deficient) or Luc+ CD4+ (nTreg cell-sufficient) BDC cells from Luc-BDC/NOD mice. We then adoptively transferred these cells separately into NOD/scid recipient mice. Trafficking and tissue localization of the transferred Luc+ BDC cells in the two cohorts were monitored until after the recipient mice that received Luc+ CD4+ CD25- (nTreg-deficient) BDC cells developed T1D (Lee et al., 2010).
We have performed initial macro imaging analyses using BLI on recipient mice during the pre-diabetic stages. Our
In addition to macro imaging studies on systemic
Based on the results discussed above, one can hypothesize that nTreg cells can exert their function by preventing the infiltration of immune cells from the periphery space into the islets. In the absence of nTreg cells, one would expect that the cellular components of invasive insulitis would be altered in comparison to those found in peri-insulitis that are maintained by the presence of nTreg cells. In addition, the change in cellular components in the islets may be due to the nTreg cells’ ability to regulate the local migration of immune cells. In studies to address these questions, we found that, compared to that in control nTreg cell-sufficient mice, the accelerated invasive insulitis in nTreg cell-deficient recipient NOD/scid mice was predominated by a population of CD11c+ dendritic cells, instead of by the adoptively transferred CD4+ BDC T cells (Lee et al., 2010). These results suggest that the presence of nTreg cells may inhibit islet-infiltration of CD11c+ dendritic cells. Indeed, using an
In summary, using
3. Conclusions and therapeutic implications
We have developed novel animal models that help investigators to perform
More importantly, these novel results demonstrate that, by combining micro and macro imaging analyses methods, the non-invasive