CD8+ T Cells Promote Pathological Angiogenesis in Ocular Neovascular Disease
Arteriosclerosis, Thrombosis, and Vascular Biology
VIEW EDITORIAL:Direct Involvement of CD8+ T Cells in Retinal Angiogenesis
Abstract
Background:
CD4+ (cluster of differentation) and CD8+ T cells are increased in the ocular fluids of patients with neovascular retinopathy, yet their role in the disease process is unknown.
Methods:
We describe how CD8+ T cells migrate into the retina and contribute to pathological angiogenesis by releasing cytokines and cytotoxic factors.
Results:
In oxygen-induced retinopathy, flow cytometry revealed the numbers of CD4+ and CD8+ T cells were increased in blood, lymphoid organs, and retina throughout the development of neovascular retinopathy. Interestingly, the depletion of CD8+ T cells but not CD4+ T cells reduced retinal neovascularization and vascular leakage. Using reporter mice expressing gfp (green fluorescence protein) in CD8+ T cells, these cells were localized near neovascular tufts in the retina, confirming that CD8+ T cells contribute to the disease. Furthermore, the adoptive transfer of CD8+ T cells deficient in TNF (tumor necrosis factor), IFNγ (interferon gamma), Prf (perforin), or GzmA/B (granzymes A/B) into immunocompetent Rag1−/− mice revealed that CD8+ T cells mediate retinal vascular disease via these factors, with TNF influencing all aspects of vascular pathology. The pathway by which CD8+ T cells migrate into the retina was identified as CXCR3 (C-X-C motif chemokine receptor 3) with the CXCR3 blockade reducing the number of CD8+ T cells within the retina and retinal vascular disease.
Conclusions:
We discovered that CXCR3 is central to the migration of CD8+ T cells into the retina as the CXCR3 blockade reduced the number of CD8+ T cells within the retina and vasculopathy. This research identified an unappreciated role for CD8+ T cells in retinal inflammation and vascular disease. Reducing CD8+ T cells via their inflammatory and recruitment pathways is a potential treatment for neovascular retinopathies.
Graphical Abstract
See accompanying editorial on page 537
Retinal vascular diseases such as retinopathy of prematurity (ROP), diabetic retinopathy, and age-related macular degeneration are significant global health challenges that continue to escalate in prevalence.1–31 These vascular diseases threaten vision and can lead to blindness due to retinal neovascularization and vascular leakage from breakdown of the blood-retinal barrier.1–3 The potent angiogenic and vascular permeability factor VEGF (vascular endothelial growth factor) is a major target for the treatment of retinal vascular disease, yet the use of anti-VEGF agents in children with ROP has raised safety concerns,4 and resistance to this therapy occurs in some patients with diabetic retinopathy and age-related macular degeneration.5,6 This situation has led to considerable interest in further understanding the mechanisms that influence the pathogenesis of retinal vascular diseases.
Inflammation is a major contributor to ROP, diabetic retinopathy, and age-related macular degeneration, with microglia, macrophage-like cells resident within the retina, having a causal role.7,8 This occurs through their release of proinflammatory factors such as TNF (tumor necrosis factor), which promote injury to the vasculature including neovascularization.9,10 In this regard, the innate immune response is well studied,11 but considerably less is known about the role of adaptive immunity. This might be due to the previous view that the retina is an immune privileged tissue and therefore impervious to immune cells produced in lymphoid tissues such as spleen and peripheral lymph nodes trafficking into the retina and the difficulties in evaluating the retina with immunological techniques. In a prior study, we utilized a robust rodent model of retinal vascular disease and ROP known as oxygen-induced retinopathy (OIR)12 to evaluate the adaptive immune system.13 We demonstrated Foxp3 (forkhead box protein 3)+CD4+CD25+ (cluster of differentation) regulatory T cells of the adaptive immune system with powerful anti-inflammatory properties can migrate from lymphoid tissues into the retina.13 Strategies increasing their abundance resulted in the deactivation of microglia, reduced VEGF levels, and neovascularization in the retina.13 Our findings raised the possibility that other cell populations of the adaptive immune system, such as effector T cells, could penetrate the retina and promote retinal vascular disease. Of interest are reports that CD4+ and CD8+ T cells are present in the vitreous fluid and epiretinal membranes of patients with proliferative diabetic retinopathy14–17 and in the retina of mice with OIR.18 However, the precise role of T cells in retinal vascular disease and their molecular mechanisms of action remain unknown.
Here, we provide compelling evidence of a pathogenic role for CD8+ T cells in retinal vascular disease. We show that the numbers of CD4+ and CD8+ T cells not only increased in the retina of OIR mice but also in the blood and lymphoid tissues. Using Rag1−/− (recombinant-activating gene 1 deficient) mice and depletion antibodies, we demonstrate that CD8+ T cells, rather than CD4+ T cells, promote neovascularization and vascular leakage in OIR. Using mice expressing gfp (green fluorescent protein) in CD8+ T cells (CD8gfp+/−), CD8+ T cells were mainly found near retinal neovessels. Flow cytometry revealed that these infiltrating CD8+ T cells displayed increased effector and memory functions. The detrimental effect of CD8+ T cells is exemplified by adoptive transfer experiments from genetically modified mice stimulating retinal vascular disease via the proinflammatory mediators TNF and IFNγ (interferon gamma) and the cytotoxic factors Prf (perforin) and GzmA/B (granzymes A/B). We identified that CXCR3 (C-X-C motif chemokine receptor 3)/IP-10 (interferon γ-induced protein 10) promotes the migration of CD8+ T cells into the OIR retina with blockade of this chemokine axis, reducing the influx of CD8+ T cells and vascular disease. These findings provide insight into the mechanisms by which CD8+ T cells influence retinal health and highlight the growing significance of the adaptive immune system in vision-threatening vascular disease.
Materials and Methods
A comprehensive description of methods can be found in the Supplemental Material. The data that support the findings of this study are available from the corresponding author upon reasonable request.
Animals
All studies were approved by the University of Melbourne Ethics Committee (number 10452) and adhered to the National Health and Medical Research Council of Australia Guidelines for the Care and Use of Animals in Scientific Research. C57BL/6J mice (JAX stock number 000664), pregnant and 6-to-8 weeks old, were obtained from the Animal Resources Centre (Perth, Western Australia). Pregnant Rag1−/− mice were purchased from the Walter and Eliza Hall Institute of Medical Research Animal Facility. We were gifted IFNγ−/− mice from Prof Paul Hertzog (Hudson Institute of Medical Research, Monash University, Clayton, Victoria, Australia), TNF−/− mice from Prof Thomas Gebhardt (Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia), and Prf−/− and GzmA/B−/− mice from Prof Joseph Trapani (Peter MacCallum Cancer Centre, Parkville, Victoria, Australia). Female CD8gfp+/− mice (JAX stock number 008766) were mated with CD8gfp−/− male mice to generate pregnant mice and heterozygous offspring. All mice were given a standard rat and mouse diet (No. SF00-100; Specialty Feeds, Perth, Australia) containing 19.6% protein and 4.4% total fat. All mice were C57BL/6J background.
Oxygen-Induced Retinopathy
The OIR procedure was performed according to our previous publications.13,19 Litters of mice were randomized to control or OIR groups, as well as treatment groups. In OIR studies, mouse pups and their nursing mothers were exposed to hyperoxia (75% oxygen) for 22 hours per day between postnatal day (P) 7 to P12 in specialized chambers that were maintained by a ProOx 110 gas regulator (Biospherix, NY) attached to medical-grade oxygen cylinders (Air Liquide, Victoria, Australia). Mice were returned to room air on P12 until P18 (Figure 1A). Age-matched controls were housed in room air (21% oxygen). As sex does not alter the development of OIR,20 both male and female mouse pups were studied at P13, P16, and P18. At the end of the study, mice were humanely killed with sodium pentobarbitone (170 mg/mL; Virbac, Peakhurst, New South Wales, Australia). OIR mice were only included in the study if they showed a consistent body weight gain in accordance with the established criteria for OIR studies.13 Body weight for each pup was recorded at the end of the study (Tables S1 and S2).
Results
CD4+ and CD8+ T Cells Are Increased in the Blood and Lymphoid Tissues of Mice With OIR
ROP is a leading cause of vision loss and blindness in preterm infants, with low gestational age, low birth weight, and supplemental oxygen therapy being the major risk factors.21–23 In addition, infection and inflammation have emerged as important contributors to ROP.22,24 The murine OIR model largely replicates the vascular pathology that develops in ROP and occurs in 2 phases over 18 days (Figure 1A). In phase I OIR, the exposure of neonatal mice to hyperoxia from P7 to P12 causes extensive vaso-obliteration. Phase I of OIR mimics the clinical setting when preterm infants receive supplemental oxygen to alleviate respiratory distress. In phase II OIR, the exposure of mice to room air for 5 days induces retinal ischemia and the excessive production of proangiogenic factors, such as VEGF, that cause marked neovascularization and vascular leakage in the retina.
To determine whether the peripheral adaptive immune system is altered in OIR and potentially contributes to retinal disease, we analyzed the abundance of CD4+ and CD8+ T cells (Figure 1B and 1C) by flow cytometry (fluorescence-activated cell sorting [FACS]) in blood, pooled lymph nodes, and spleen during the neovascular phase II of OIR at P13, P16, and P18. Data were expressed as the percentage of total lymphocytes for CD4+ and CD8+ T cells. CD4+ T cells were increased in OIR in the blood and spleen at P13 and P16 compared with age-matched controls (Figure 1B). CD8+ T cells were increased in spleen at all time points (P13, P16, and P18) and in lymph nodes at P16 compared with controls (Figure 1C). Interestingly, at P13, when retinal ischemia is most acute between phases I and II of OIR, CD8+ T cells were transiently reduced in lymph nodes compared with controls (Figure 1C), which coincides with the increased number of regulatory T cells reported in our previous study.13
CD4+ and CD8+ T Cells Increased in the Retina of Mice With OIR
The next step was to determine whether CD4+ and CD8+ T cells migrated into the retina. To accurately identify and quantitate T cells in retina, we developed a novel immunotyping method using FACS. Consistent with other studies in neural tissues,25 CD4+ and CD8+ T cells were found within the CD45hi inflammatory cell population (Figure 2A through 2D). Due to the increased number of CD4+ and CD8+ T cells in blood and lymphoid organs at P16, we quantified their number in the retina at this time point (Figure 2A through 2G). The percentages of both CD4+ and CD8+ T cells within the lymphocyte gate increased in the retina of OIR mice compared with controls (Figure 2G and 2H).
Rag1−/− Mice With OIR Have Reduced Retinal Vascular Disease
To investigate T cells in OIR, we first evaluated Rag1−/− mice that lack both T and B cells.26 In Rag1−/− control mice, retinal vascularization appeared normal and similar to their genetic controls, C57BL/6J wild-type mice (Figure 3A). As expected in wild-type OIR mice, retinal neovascularization developed13 (Figure 3A and 3B) but was about 50% lower in Rag1−/− mice with OIR (Figure 3B). In wild-type OIR mice, retinal vascular leakage was increased compared with controls and reduced in Rag1−/− mice with OIR (Figure 3C). Consistent with these data, in wild-type OIR mice, VEGF protein and mRNA levels in retina were increased compared with controls and reduced in Rag1−/− mice with OIR compared with wild-type OIR mice (Figure 3D and 3E). These findings suggest a pathogenic role for T cells in OIR, but we cannot exclude the possibility that the absence of B cells in Rag1−/− mice may have influenced retinal vascular disease.
CD8α- and CD8β-, but not CD4-Depleting Antibodies, Reduced Retinal Vascular Disease in Mice With OIR
To interrogate the specific involvement of CD4+ and CD8+ T cells in OIR, C57BL/6J mice with OIR were depleted of CD4+ and CD8+ T cells by utilizing monoclonal antibodies. Mice were given a single intraperitoneal injection of depletion antibodies (100 μg per mouse) at P10 before phase II of OIR. Administration of the CD4-specific monoclonal antibody (clone YTS191) depleted >95% of CD4+ T cells in the blood and lymphoid tissues of mice with OIR compared with mice treated with the isotype IgG1 control antibody (Figure S1A through S1F). CD8+ T cells were depleted using a CD8α-specific antibody, clone YTS169, which depleted >95% of CD8+ T cells in the blood and lymphoid tissues of mice with OIR (Figure S1G). Because CD8α antibodies also deplete dendritic cells expressing CD11c and CD8α,27 a CD8β-depleting antibody (clone 5.3-58) that spares CD8+ dendritic cells was used to further assess the involvement of CD8+ T cells. In the CD8β antibody–treated mice, CD8+ T cells were reduced by 95% in the blood, spleen, and lymph nodes (Figure S1H).
The CD8α but not the CD4 depletion antibody reduced retinal neovascularization by about 30% (Figure 4A and 4B), vascular leakage by >50% (Figure 4C), and VEGF protein levels (Figure 4D) in mice with OIR compared with mice treated with the IgG2a isotype control antibody. The vascular disease induced by CD8+ T cells was confirmed in mice administered the CD8β-depleting antibody, which reduced retinal neovascularization by 45%, vascular leakage by 50%, and VEGF protein levels (Figure S2A through S2D) compared with mice treated with the IgG1 isotype control antibody. Consistent with the inability of CD4+ T-cell depletion to lessen retinal neovascularization (Figure 4A and 4B), CD4+ T-cell depletion did not reduce retinal vascular leakage (Figure 4C) and VEGF protein levels (Figure 4D) in mice with OIR.
An important next step was to evaluate the location and activation status of CD8+ T cells in the retina. To locate CD8+ T cells accurately, we used transgenic CD8gfp+/− mice that express a GFP specifically in CD8+ T cells. We first confirmed the expression of GFP by CD8+ T cells in CD8gfp+/− mice using FACS and found ≈90% of CD8+ T cells positive for GFP in lymph nodes and spleen (Figure S3). GFP+CD8+ T cells in the retina were then evaluated by confocal microscopy imaging. In controls, CD8+ T cells were rarely found in the retina (Figure 5A) but in OIR mice, were located near areas of retinal neovascularization (Figure 5B) and were increased compared with controls (Figure 5C), data consistent with the FACS results (Figure 2H). To characterize the activation status of infiltrating CD8+ T cells, we performed FACS with CD44 and CD62L antibodies (Figure 5D through 5G). CD8+ T cells were predominantly naive in the control retina (Figure 5D). In OIR, this proportion of naive CD8+ T cells remained unchanged in the retina (Figure 5E). However, effector (CD44+CD62L−; Figure 5F) and memory (CD44+CD62L+; Figure 5G) CD8+ T cells increased with OIR, suggesting that there are more activated CD8+ T cells causing vascular injury.
CD8+ T Cells Deficient in Proinflammatory and Cytotoxic Factors Are Less Able to Induce Retinal Vascular Disease in OIR
We investigated the mechanisms by which CD8+ T cells promote injury to the retinal vasculature. Activated CD8+ T cells are an important source of proinflammatory factors, such as TNF and IFNγ in inflammation-induced tissue injury.28 Furthermore, CD8+ T cells, upon activation, become cytotoxic T cells and cause cell lysis by secreting Prf and granzymes, resulting in tissue inflammation.29 To determine whether CD8+ T cells cause retinal vascular damage via these proinflammatory and cytotoxic factors, we adoptively transferred 106 CD8+ T cells from IFNγ−/−, TNF−/−, Prf−/−, and GzmA/B−/− mice into lymphocyte-deficient Rag1−/− mice with OIR. We first confirmed that the adoptively transferred CD8+ T cells were able to repopulate peripheral lymphoid tissues of Rag1−/− OIR (Figure S4).
In Rag1−/− mice with OIR, adoptively transferred CD8+ T cells from wild-type mice increased neovascularization, vascular leakage, and VEGF protein levels in retina compared with mice administered vehicle (Figure 6A through 6D), further confirming the pathogenic role of CD8+ T cells in retinal vascular pathology. Compared with wild-type CD8+ T cells, CD8+ T cells deficient in IFNγ, TNF, Prf, and GzmA/B were less able to increase retinal neovascularization (Figure 6A and 6B) and VEGF protein (Figure 6C) and mRNA levels (Figure S5A) in Rag1−/− mice with OIR. Retinal vascular leakage was only reduced significantly in TNF−/− CD8+ T-cell recipient mice (Figure 6D). To determine whether the depletion of specific factors from CD8+ T cells altered the expression of inflammatory mediators implicated in the development of OIR, we measured TNF, ICAM-1 (intercellular adhesion molecule-1), and MCP-1 (monocyte chemoattractant protein-1) levels in retina. Consistent with our findings that TNF is the main factor produced by CD8+ T cells that influences retinal vascular disease in OIR, only TNF-deficient CD8+ T cells reduced TNF and ICAM-1 mRNA levels and MCP-1 protein levels in retina of Rag1−/− mice with OIR (Figure S5B through S5D). The administration of IFNγ−/− CD8+ T cells reduced MCP-1 protein levels, and both Prf−/− and GzmA/B−/− CD8+ T cells reduced TNF and ICAM-1 mRNA levels in the retina of Rag1−/− OIR mice compared with Rag1−/− OIR mice administered wild-type CD8+ T cells (Figure S5B through S5D).
CXCR3/IP-10 Axis in CD8+ T Cells Is Increased in OIR
To investigate the mechanism whereby CD8+ T cells are recruited into the retina, we measured a range of chemotactic cytokines and chemokines using a multiplex bead assay. We measured the expression of 13 chemokines in the acute phase II of OIR at P13. Among the 13 chemokines detected, the levels of IP-10, thymus, and activation-regulated chemokine/CCL (C-C motif ligand) 17, macrophage inflammatory protein-1b/CCL4, lipopolysaccharide-induced CXC chemokine/CXCL5 (C-X-C chemokine 5), monocyte-derived chemokine/CCL22, and eotaxin/CCL11 were significantly increased in the retina of mice with OIR (Figure 7A).
The chemokine IP-10/CXCL10 (C-X-C motif chemokine ligand 10) is of particular interest as its receptor CXCR3 is preferentially expressed by activated CD8+ T cells and plays a vital role in their trafficking into the inflamed site.30,31 Therefore, we measured the expression of CXCR3 on CD8+ T cells in the blood and retina of OIR mice. As shown in Figure 7B through 7G, the number of circulating CXCR3+CD8+ T cells was increased in OIR compared with control mice, suggesting that CD8+ T cells utilize the CXCR3/IP-10 chemokine-receptor axis as a gateway to infiltrate the retina. We further confirmed this finding by showing that the number of CXCR3+CD8+ T cells increased in the retina of OIR mice compared with controls (Figure 7E through 7G). Interestingly, we found that the number of CD69+CD8+ T cells was increased in the blood and lymphoid tissues of mice with OIR (Figure S6), further suggesting that CD8+ T cells are more activated in OIR.
CXCR3 Blockade Reduces CD8+ T-Cell Infiltration and Attenuates Retinal Neovascularization in OIR
To demonstrate that CD8+ T cells were recruited into the retina via CXCR3/IP-10, we blocked CXCR3 using a monoclonal antibody (clone IC6).30 We found that the infiltration of CD8+ T cells into the retina was reduced by 50% in CXCR3 antibody–treated mice compared with the isotype control antibody–treated mice (Figure 8A through 8C). CD4+ T cells were unaffected by the antibody treatment, further confirming the specificity of the CXCR3/IP-10 axis for CD8+ T cells in OIR (Figure 8D). We then used CD8gfp+/− mice to quantitate the number of infiltrating CD8+ T cells in the retina by confocal imaging (Figure 8E and 8F) and confirmed the CXCR3 blocking antibody reduced the number of CD8+ T cells within the retina of OIR mice by ≈40% (Figure 8G). Importantly, neovascularization, vascular leakage, and VEGF protein levels were all reduced in the retina of OIR mice treated with the anti-CXCR3 antibody (Figure 8H through 8K), indicating the importance of the CXCR3/IP-10 pathway in the recruitment of CD8+ T cells into the retina and their subsequent induction of retinal vascular disease.
Discussion
Here, we revealed a novel pathogenic role for CD8+ T cells, supporting their involvement in retinal vascular disease. CD8+ T cells are found in the ocular fluids of patients with severe retinal vascular disease such as proliferative diabetic retinopathy,14,15,32 but their migration into retinal tissue and regulation of vascular injury is unknown. The mechanisms by which CD8+ T cells proliferate in the lymphoid organs of OIR mice are unclear and may be related to tissue hypoxia, which induces necrosis or apoptosis of cells and subsequently stimulates the recruitment and activation of CD8+ T cells.33 Such CD8+ T-cell infiltration and activation occurs in ischemic conditions, such as atherosclerosis27,34 and myocardial infarction.35 The presence of CD8+ T cells within the retina of CD8 reporter mice and their increased abundance in OIR is an indication that CD8+ T cells have the capacity to migrate into the retina. Both the depletion and adoptive transfer of CD8+ T cells influenced vascular injury, supporting a pathogenic role for CD8+ T cells in the vision-threatening events of retinal neovascularization and vascular leakage.
Our study identified diverse mechanisms by which CD8+ T cells promote retinal vascular disease. CD8+ T cells secrete the potent proinflammatory factors TNF and IFNγ, as well as the cytotoxic factors Prf and GzmA/B, that are of potential significance to retinal vascular disease.36–38 In comparison to the vascular damage seen with the transfer of wild-type CD8+ T cells, IFNγ-, TNF-, Prf-, and GzmA/B-deficient CD8+ T cells were less able to induce neovascularization and VEGF expression in the retina of lymphocyte-deficient OIR mice. The transfer of TNF-deficient CD8+ T cells was the most effective in reducing vascular disease in OIR, suggesting TNF is a main mechanism by which CD8+ T cells mediate injury to the retinal vasculature. There is substantial evidence that TNF is involved in the development of vascular retinopathies with elevated levels found in the serum of children with ROP39,40 and people with diabetic retinopathy41,42 and the ability of TNF to stimulate retinal neovascularization and breakdown of the blood-retinal barrier.43,44 Traditionally, activated microglia are viewed as the primary source of TNF in the retina.7,45,46 Our data revealed CD8+ T cells are a major contributor to TNF’s mediation of retinal vascular disease in OIR.
We demonstrated that in addition to TNF, other factors secreted from CD8+ T cells influence retinal vascular disease, namely, IFNγ, Prf, and GzmA/B, but we speculate this occurs by different pathways. In terms of IFNγ, this cytokine may influence ocular angiogenesis by augmenting VEGF levels. In human retinal pigment epithelial cells, IFNγ through the PI3K (phosphoinositide 3-kinase)/mTOR (mammalian target of rapamycin) pathway increases VEGF expression and possibly choroidal angiogenesis.47 IFNγ can also influence the proliferation of retinal microglia, which release factors that promote local inflammation and angiogenesis such as VEGF, TNF, IL (interleukin)-1, and IL-6.7,45,46 A well-recognized function of CD8+ T cells is their induction of cytotoxicity primarily by Prf and GzmA/B.28 Prf released from CD8+ T cells forms a pore in the membrane of the target cell allowing granzymes to enter the cytoplasm and cause apoptosis.48 However, these cytotoxic factors can influence events unrelated to cell death, including vascular injury.49,50 For instance, Prf-deficient mice are resistant to vascular permeability in the central nervous system.51 Granzyme B promotes the cleavage and activation of extracellular sequestered VEGF,49,52 which induces the growth of capillaries and vascular permeability.49,50 Our data are consistent with these properties of Prf- and GzmA/B-deficient CD8+ T cells reducing vascular disease and inflammation in the OIR retina, albeit vascular leakage was not altered for reasons unknown.
The pathway mediating the recruitment of CD8+ T cells into the retina is unknown. We specifically identified the chemokine IP-10 to be increased in the OIR retina and involved in the recruitment of CD8+ T cells via the receptor for IP-10, CXCR3. This chemokine is highly expressed on T cells within peripheral tissues in autoimmune and inflammatory conditions,30,31 and its absence from CD8+ T cells ameliorates immune-related tissue damage.53 Our findings that CXCR3 blockade reduced the infiltration of CD8+ T cells into the retina and retinal inflammation indicate a proinflammatory role for CXCR3 in vascular retinopathy. Further, our data are consistent with the activation of CD8+ T cells being linked to the upregulation of CXCR3,31 as CD8+ T cells in the blood and lymphoid organs of OIR mice had increased expression levels of activation markers, such as CD69.
To better understand the composition of CD8+ T cells in the retina, we evaluated their memory phenotypes by flow cytometry. In OIR, effector and memory T-cell frequencies were significantly expanded in the retina from 0% to ≈5% and ≈20%, respectively. In general, OIR results in an overall expansion in the CD44+ frequency in the retina, suggesting that there are more activated CD8+ T cells. Previous studies have demonstrated that CXCR3 facilitates the migration of effector CD8+ T cells to inflamed sites.54,55 In line with this, we found increased numbers of CXCR3+CD8+ T cells in the retina, suggesting a possible involvement of CXCR3 in the recruitment of effector CD8+ T cells in OIR. A limitation of the study is that it is unclear whether the activation of CD8+ T cells is mediated by an antigen. It is possible that molecular mimicry or bystander activation could play a role in the activation of CD8+ T cells in our model.56,57 Further studies are warranted to identify whether an antigen is involved in the expansion of effector and memory CD8+ T cells or whether they are antigen-independent innate memory T cells in OIR.
We acknowledge that the mechanisms by which CD8+ T cells penetrate into the retina may occur via other chemokines, such as CXCL9 (monokine induced by IFNγ), which also binds to CXCR3 and is produced by retinal microglia under hypoxic conditions.44 CXCR3 antagonists are an active area of research for the treatment of inflammatory diseases including multiple sclerosis, inflammatory bowel disease, and atherosclerosis,31,58 and it is noteworthy that vitreous levels of IP-10 are increased in people with diabetic retinopathy.59 Our finding that CD8+ T cells migrated to the retina via CXCR3 and IP-10 to cause retinal vascular disease provides insights into novel therapeutics that target chemokines and their receptors.
In summary, we have identified that CD8+ T cells via their release of TNF, IFNγ, Prf, and GzmA/B have a pathogenic role in retinal vascular disease. Our work highlights the potential of inhibiting the recruitment or the effector functions of CD8+ T cells as new treatment strategies for vision-threatening diseases such as ROP.
Article Information
Supplemental Material
Supplemental Materials and Methods
Figures S1–S6
Tables S1 and S2
Major Resources Table
Acknowledgments
The authors thank Dr Abhirup Jayasimhan, Dr Amit Joglekar, and Varaporn Suphapimol from the Department of Anatomy and Physiology at the University of Melbourne for their technical assistance. We thank the Biological Optical Microscopy Platform and the Flow Cytometry Facility in the Melbourne Brain Centre at the University of Melbourne.
Footnote
Nonstandard Abbreviations and Acronyms
- CCL
- C-C motif ligand
- FACS
- fluorescence-activated cell sorting
- Foxp3
- forkhead box protein 3
- GFP
- green fluorescence protein
- GzmA/B
- granzymes A/B
- ICAM-1
- intercellular adhesion molecule-1
- IFNγ
- interferon gamma
- IL
- interleukin
- MCP-1
- monocyte chemoattractant protein-1
- mTOR
- mammalian target of rapamycin
- OIR
- oxygen-induced retinopathy
- P
- postnatal day
- PI3K
- phosphoinositide 3-kinase
- Prf
- perforin
- Rag1−/−
- recombinant-activating gene 1 deficient
- ROP
- retinopathy of prematurity
- TNF
- tumor necrosis factor
- VEGF
- vascular endothelial growth factor
Supplemental Material
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- Download
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References
1.
Hellström A, Smith LEH, Dammann O. Retinopathy of prematurity. Lancet. 2013;382:1445–1457. doi: 10.1016/S0140-6736(13)60178-6
2.
Cheung N, Mitchell P, Wong TY. Diabetic retinopathy. Lancet. 2010;376:124–136. doi: 10.1016/S0140-6736(09)62124-3
3.
Das A, McGuire PG. Retinal and choroidal angiogenesis: pathophysiology and strategies for inhibition. Prog Retin Eye Res. 2003;22:721–748. doi: 10.1016/j.preteyeres.2003.08.001
4.
Enríquez AB, Avery RL, Baumal CR. Update on anti-vascular endothelial growth factor safety for retinopathy of prematurity. Asia Pac J Ophthalmol (Phila). 2020;9:358–368. doi: 10.1097/APO.0000000000000302
5.
Simó R, Hernández C. Novel approaches for treating diabetic retinopathy based on recent pathogenic evidence. Prog Retin Eye Res. 2015;48:160–180. doi: 10.1016/j.preteyeres.2015.04.003
6.
Kokame GT, deCarlo TE, Kaneko KN, Omizo JN, Lian R. Anti-vascular endothelial growth factor resistance in exudative macular degeneration and polypoidal choroidal vasculopathy. Ophthalmol Retina. 2019;3:744–752. doi: 10.1016/j.oret.2019.04.018
7.
Karlstetter M, Scholz R, Rutar M, Wong WT, Provis JM, Langmann T. Retinal microglia: just bystander or target for therapy?. Prog Retin Eye Res. 2015;45:30–57. doi: 10.1016/j.preteyeres.2014.11.004
8.
Tang J, Kern TS. Inflammation in diabetic retinopathy. Prog Retin Eye Res. 2011;30:343–358. doi: 10.1016/j.preteyeres.2011.05.002
9.
Deliyanti D, Armani R, Casely D, Figgett WA, Agrotis A, Wilkinson-Berka JL. Retinal vasculopathy is reduced by dietary salt restriction: involvement of glia, ENaCα, and the renin-angiotensin-aldosterone system. Arterioscler Thromb Vasc Biol. 2014;34:2033–2041. doi: 10.1161/ATVBAHA.114.303792
10.
Grigsby JG, Cardona SM, Pouw CE, Muniz A, Mendiola AS, Tsin ATC, Allen DM, Cardona AE. The role of microglia in diabetic retinopathy. J Ophthalmology. 2014;2014:705783–705783. doi: 10.1155/2014/705783
11.
Xu H, Chen M. Diabetic retinopathy and dysregulated innate immunity. Vis Res. 2017;139:39–46. doi: 10.1016/j.visres.2017.04.013
12.
Stahl A, Connor KM, Sapieha P, Chen J, Dennison RJ, Krah NM, Seaward MR, Willett KL, Aderman CM, Guerin KI, et al. The mouse retina as an angiogenesis model. Invest Ophthalmol Vis Sci. 2010;51:2813–2826. doi: 10.1167/iovs.10-5176
13.
Deliyanti D, Talia DM, Zhu T, Maxwell MJ, Agrotis A, Jerome JR, Hargreaves EM, Gerondakis S, Hibbs ML, Mackay F, et al. Foxp3+ Tregs are recruited to the retina to repair pathological angiogenesis. Nat Commun. 2017;8:748. doi: 10.1038/s41467-017-00751-w
14.
Urbančič M, Kloboves Prevodnik V, Petrovič D, Globočnik Petrovič M. A flow cytometric analysis of vitreous inflammatory cells in patients with proliferative diabetic retinopathy. Biomed Res Int. 2013;2013:251528. doi: 10.1155/2013/251528
15.
Cantón A, Martinez-Cáceres EM, Hernández C, Espejo C, García-Arumí J, Simó R. CD4-CD8 and CD28 expression in T cells infiltrating the vitreous fluidin patients with proliferative diabetic retinopathy: a flow cytometric analysis. Arch Ophthalmol. 2004;122:743–749. doi: 10.1001/archopht.122.5.743
16.
Charteris DG, Hiscott P, Grierson I, Lightman SL. Proliferative vitreoretinopathy. Lymphocytes in epiretinal membranes. Ophthalmology. 1992;99:1364–1367. doi: 10.1016/s0161-6420(92)31793-2
17.
Limb GA, Franks WA, Munasinghe KR, Chignell AH, Dumonde DC. Proliferative vitreoretinopathy: an examination of the involvement of lymphocytes, adhesion molecules and HLA-DR antigens. Graefes Arch Clin Exp Ophthalmol. 1993;231:331–336. doi: 10.1007/BF00919029
18.
Zhou Y, Yoshida S, Kubo Y, Kobayashi Y, Nakama T, Yamaguchi M, Ishikawa K, Nakao S, Ikeda Y, Ishibashi T, et al. Interleukin-12 inhibits pathological neovascularization in mouse model of oxygen-induced retinopathy. Sci Rep. 2016;6:28140. doi: 10.1038/srep28140
19.
Wilkinson-Berka JL, Deliyanti D, Rana I, Miller AG, Agrotis A, Armani R, Szyndralewiez C, Wingler K, Touyz RM, Cooper ME, et al. NADPH oxidase, NOX1, mediates vascular injury in ischemic retinopathy. Antioxid Redox Signal. 2014;20:2726–2740. doi: 10.1089/ars.2013.5357
20.
Higgins RD, Yan Y, Tadesse M, Yossuck P. Lack of effect of gender on retinopathy in the mouse. Clin Exp Ophthalmol. 2001;29:323–326. doi: 10.1046/j.1442-9071.2001.00441.x
21.
Darlow BA, Hutchinson JL, Henderson-Smart DJ, Donoghue DA, Simpson JM, Evans NJ; Australian and New Zealand Neonatal Network. Prenatal risk factors for severe retinopathy of prematurity among very preterm infants of the Australian and New Zealand Neonatal Network. Pediatrics. 2005;115:990–996. doi: 10.1542/peds.2004-1309
22.
Chen M, Citil A, McCabe F, Leicht KM, Fiascone J, Dammann CE, Dammann O. Infection, oxygen, and immaturity: interacting risk factors for retinopathy of prematurity. Neonatology. 2011;99:125–132. doi: 10.1159/000312821
23.
Campbell K. Intensive oxygen therapy as a possible cause of retrolental fibroplasia; a clinical approach. Med J Aust. 1951;2:48–50.
24.
Rivera JC, Holm M, Austeng D, Morken TS, Zhou T, Beaudry-Richard A, Sierra EM, Dammann O, Chemtob S. Retinopathy of prematurity: inflammation, choroidal degeneration, and novel promising therapeutic strategies. J Neuroinflammation. 2017;14:165. doi: 10.1186/s12974-017-0943-1
25.
Wagner CA, Roqué PJ, Mileur TR, Liggitt D, Goverman JM. Myelin-specific CD8+ T cells exacerbate brain inflammation in CNS autoimmunity. J Clin Investig. 2020;130:203–213. doi: 10.1172/JCI132531
26.
Mombaerts P, Iacomini J, Johnson RS, Herrup K, Tonegawa S, Papaioannou VE. RAG-1-deficient mice have no mature B and T lymphocytes. Cell. 1992;68:869–877. doi: 10.1016/0092-8674(92)90030-g
27.
Kyaw T, Winship A, Tay C, Kanellakis P, Hosseini H, Cao A, Li P, Tipping P, Bobik A, Toh BH. Cytotoxic and proinflammatory CD8+ T lymphocytes promote development of vulnerable atherosclerotic plaques in apoE-deficient mice. Circulation. 2013;127:1028–1039. doi: 10.1161/CIRCULATIONAHA.112.001347
28.
Smyth MJ, Kelly JM, Sutton VR, Davis JE, Browne KA, Sayers TJ, Trapani JA. Unlocking the secrets of cytotoxic granule proteins. J Leukoc Biol. 2001;70:18–29. doi: 10.1189/jlb.70.1.18
29.
Mittrücker HW, Visekruna A, Huber M. Heterogeneity in the differentiation and function of CD8⁺ T cells. Arch Immunol Ther Exp (Warsz). 2014;62:449–458. doi: 10.1007/s00005-014-0293-y
30.
Qin S, Rottman JB, Myers P, Kassam N, Weinblatt M, Loetscher M, Koch AE, Moser B, Mackay CR. The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions. J Clin Investig. 1998;101:746–754. doi: 10.1172/jci1422
31.
Groom JR, Luster AD. CXCR3 in T cell function. Exp Cell Res. 2011;317:620–631. doi: 10.1016/j.yexcr.2010.12.017
32.
Urbančič M, Štunf S, Milutinović Živin A, Petrovič D, Globočnik Petrovič M. Epiretinal membrane inflammatory cell density might reflect the activity of proliferative diabetic retinopathy. Invest Ophthalmol Vis Sci. 2014;55:8576–8582. doi: 10.1167/iovs.13-13634
33.
Feig C, Peter ME. How apoptosis got the immune system in shape. Eur J Immunol. 2007;37:S61–S70. doi: 10.1002/eji.200737462
34.
Cochain C, Zernecke A. Protective and pathogenic roles of CD8+ T cells in atherosclerosis. Basic Res Cardiol. 2016;111:71. doi: 10.1007/s00395-016-0589-7
35.
Santos-Zas I, Lemarié J, Zlatanova I, Cachanado M, Seghezzi J-C, Benamer H, Goube P, Vandestienne M, Cohen R, et al. Cytotoxic CD8+ T cells promote granzyme B-dependent adverse post-ischemic cardiac remodeling. Nat Commun. 2021;12:1483. doi: 10.1038/s41467-021-21737-9
36.
Sato T, Kusaka S, Shimojo H, Fujikado T. Simultaneous analyses of vitreous levels of 27 cytokines in eyes with retinopathy of prematurity. Ophthalmology. 2009;116:2165–2169. doi: 10.1016/j.ophtha.2009.04.026
37.
Chang J, Eggenhuizen P, O’Sullivan KM, Alikhan MA, Holdsworth SR, Ooi JD, Kitching AR. CD8+ T cells effect glomerular injury in experimental anti-myeloperoxidase GN. J Am Soc Nephrol. 2017;28:47–55. doi: 10.1681/ASN.2015121356
38.
Ma F, Feng J, Zhang C, Li Y, Qi G, Li H, Wu Y, Fu Y, Zhao Y, Chen H, et al. The requirement of CD8+ T cells to initiate and augment acute cardiac inflammatory response to high blood pressure. J Immunol. 2014;192:3365–3373. doi: 10.4049/jimmunol.1301522
39.
Sood BG, Madan A, Saha S, Schendel D, Thorsen P, Skogstrand K, Hougaard D, Shankaran S, Carlo W; NICHD Neonatal Research Network. Perinatal systemic inflammatory response syndrome and retinopathy of prematurity. Pediatric Res. 2010;67:394–400. doi: 10.1203/PDR.0b013e3181d01a36
40.
Yu H, Yuan L, Zou Y, Peng L, Wang Y, Li T, Tang S. Serum concentrations of cytokines in infants with retinopathy of prematurity. APMIS. 2014;122:818–823. doi: 10.1111/apm.12223
41.
Gustavsson C, Agardh E, Bengtsson B, Agardh CD. TNF-alpha is an independent serum marker for proliferative retinopathy in type 1 diabetic patients. J Diabetes Complications. 2008;22:309–316. doi: 10.1016/j.jdiacomp.2007.03.001
42.
Huang H, Gandhi JK, Zhong X, Wei Y, Gong J, Duh EJ, Vinores SA. TNFalpha is required for late BRB breakdown in diabetic retinopathy, and its inhibition prevents leukostasis and protects vessels and neurons from apoptosis. Invest Ophthalmol Vis Sci. 2011;52:1336–1344. doi: 10.1167/iovs.10-5768
43.
Gardiner TA, Gibson DS, de Gooyer TE, de la Cruz VF, McDonald DM, Stitt AW. Inhibition of tumor necrosis factor-alpha improves physiological angiogenesis and reduces pathological neovascularization in ischemic retinopathy. Am J Pathol. 2005;166:637–644. doi: 10.1016/s0002-9440(10)62284-5
44.
Kociok N, Radetzky S, Krohne TU, Gavranic C, Joussen AM. Pathological but not physiological retinal neovascularization is altered in TNF-Rp55-receptor-deficient mice. Invest Ophthalmol Vis Sci. 2006;47:5057–5065. doi: 10.1167/iovs.06-0407
45.
Rana I, Suphapimol V, Jerome JR, Talia DM, Deliyanti D, Wilkinson-Berka JL. Angiotensin II and aldosterone activate retinal microglia. Exp Eye Res. 2020;191:107902. doi: 10.1016/j.exer.2019.107902
46.
Deliyanti D, Wilkinson-Berka JL. Inhibition of NOX1/4 with GKT137831: a potential novel treatment to attenuate neuroglial cell inflammation in the retina. J Neuroinflammation. 2015;12:136. doi: 10.1186/s12974-015-0363-z
47.
Liu B, Faia L, Hu M, Nussenblatt RB. Pro-angiogenic effect of IFN-gamma is dependent on the PI3K/mTOR/translational pathway in human retinal pigmented epithelial cells. Mol Vis. 2010;16:184–193.
48.
Trapani JA, Smyth MJ. Functional significance of the perforin/granzyme cell death pathway. Nat Rev Immunol. 2002;2:735–747. doi: 10.1038/nri911
49.
Hendel A, Granville DJ. Granzyme B cleavage of fibronectin disrupts endothelial cell adhesion, migration and capillary tube formation. Matrix Biol. 2013;32:14–22. doi: 10.1016/j.matbio.2012.11.013
50.
Hendel A, Hsu I, Granville DJ. Granzyme B releases vascular endothelial growth factor from extracellular matrix and induces vascular permeability. Lab Invest. 2014;94:716–725. doi: 10.1038/labinvest.2014.62
51.
Suidan GL, McDole JR, Chen Y, Pirko I, Johnson AJ. Induction of blood brain barrier tight junction protein alterations by CD8 T cells. PLoS One. 2008;3:e3037. doi: 10.1371/journal.pone.0003037
52.
Shen Y, Cheng F, Sharma M, Merkulova Y, Raithatha SA, Parkinson LG, Zhao H, Westendorf K, Bohunek L, Bozin T, et al. Granzyme B deficiency protects against angiotensin II-induced cardiac fibrosis. Am J Pathol. 2016;186:87–100. doi: 10.1016/j.ajpath.2015.09.010
53.
Christensen JE, de Lemos C, Moos T, Christensen JP, Thomsen AR. CXCL10 is the key ligand for CXCR3 on CD8+ effector T cells Involved in immune surveillance of the lymphocytic choriomeningitis virus-infected central nervous system. J Immunol. 2006;176:4235–4243. doi: 10.4049/jimmunol.176.7.4235
54.
Kurachi M, Kurachi J, Suenaga F, Tsukui T, Abe J, Ueha S, Tomura M, Sugihara K, Takamura S, Kakimi K, et al. Chemokine receptor CXCR3 facilitates CD8+ T cell differentiation into short-lived effector cells leading to memory degeneration. J Exp Med. 2011;208:1605–1620. doi: 10.1084/jem.20102101
55.
Hu JK, Kagari T, Clingan JM, Matloubian M. Expression of chemokine receptor CXCR3 on T cells affects the balance between effector and memory CD8 T-cell generation. Proc Natl Acad Sci USA. 2011;108:E118–E127. doi: 10.1073/pnas.1101881108
56.
Sprent J, Surh CD. Normal T cell homeostasis: the conversion of naive cells into memory-phenotype cells. Nat Immunol. 2011;12:478–484. doi: 10.1038/ni.2018
57.
Jameson SC, Lee YJ, Hogquist KA. Innate memory T cells. Adv Immunol. 2015;126:173–213. doi: 10.1016/bs.ai.2014.12.001
58.
Van Raemdonck K, Van den Steen PE, Liekens S, Van Damme J, Struyf S. CXCR3 ligands in disease and therapy. Cytokine Growth Factor Rev. 2015;26:311–327. doi: 10.1016/j.cytogfr.2014.11.009
59.
Elner SG, Strieter R, Bian ZM, Kunkel S, Mokhtarzaden L, Johnson M, Lukacs N, Elner VM. Interferon-induced protein 10 and interleukin 8: C-X-C chemokines present in proliferative diabetic retinopathy. Arch Ophthalmol. 1998;116:1597–1601. doi: 10.1001/archopht.116.12.1597
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Received: 27 June 2022
Accepted: 1 February 2023
Published online: 16 February 2023
Published in print: April 2023
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This work was supported by the National Health and Medical Research Council of Australia to J. Wilkinson-Berka (APP1181462) and a Juvenile Diabetes Research Foundation postdoctoral award (3-PDF-2017-376-A-N) to D. Deliyanti.
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