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Research Article
Originally Published 16 February 2023
Free Accessfeatured article

CD8+ T Cells Promote Pathological Angiogenesis in Ocular Neovascular Disease

Arteriosclerosis, Thrombosis, and Vascular Biology

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

Highlights

CD8+ (cluster of differentation) T cells promote retinal inflammation and vascular injury via inflammatory and cytotoxic factors, such as TNF (tumor necrosis factor), IFN- γ (interferon gamma), perforin, and granzymes.
CD8+ T cells migrate to the retina via the CXCR3 (C-X-C motif chemokine receptor 3)/IP-10 (interferon γ-induced protein 10) axis to cause vascular pathology.
Reducing CD8+ T cells via their inflammatory and recruitment pathways is a potential treatment for neovascular retinopathies such as retinopathy of prematurity.
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).
Figure 1. CD4+ (cluster of differentation) and CD8+ T cells in blood and lymphoid tissues in the neovascular phase II of oxygen-induced retinopathy (OIR). A, C57BL/6J mice were studied at postnatal day (P) 13, P16, and P18 and comparisons made to age-matched room air controls. Fluorescence-activated cell sorting of blood, pooled lymph nodes, and spleen for (B) CD4+ and (C) CD8+ T cells. P values were calculated using parametric unpaired t tests with Welch correction (B and C). n=11 to 14 mice per group from 3 to 4 independent experiments. Values are mean±SD.

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).
Figure 2. CD4+ (cluster of differentation) and CD8+ T cells are increased in retina in the neovascular phase II of oxygen-induced retinopathy (OIR). A through D, Flow cytometry gating strategy for CD4+ and CD8+ T cells in retina. Forward versus side scatter gate (A), single cells (B), live cells (C), and CD45hiCD3+ cells (D). Flow cytometry dot plots of CD4+ T cells and CD8+ T cells in retina from C57BL/6J (E) room air control and (F) OIR mice at postnatal day 16. CD4+ T cells (G) and CD8+ T cell numbers (H). P values were calculated using parametric unpaired t tests with Welch correction (G and H). Values are mean±SD. FSC-A indicates forward scatter area; FSC-H, forward scatter height; and SSC-A, side scatter area.

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.
Figure 3. Rag1−/− (recombinant-activating gene 1 deficient) mice with oxygen-induced retinopathy (OIR) have reduced retinal vascular disease. A, Representative flat mounts of retina at postnatal day 18 from Rag1−/− mice stained with FITC (fluorescein isothiocyanate)-conjugated isolectin B4 to identify the vasculature (green). The 2 top images show normal vascularization in the entire retina from control WT (wild type) C57BL/6J mice and Rag1−/− mice. Yellow boxes in the OIR panel are presented at higher magnification in the second bottom panel to show neovascularization (arrowheads), which has been pseudocolorized in red in the lowest panel. Asterisks denote vaso-obliteration. Scale bars, 0.125 mm. B, Retinal neovascularization in OIR mice. n=9 mice per group from 3 independent experiments. C, Retinal vascular leakage (albumin ELISA) in OIR and room air control mice. D, VEGF (vascular endothelial growth factor) protein and (E) VEGF mRNA levels in OIR and room air control mice. n=4 to 7 mice per group. P values were calculated by the parametric unpaired t test with Welch correction (B) and a 1-way ANOVA followed by the Holm-Sidak multiple comparisons test (C–E). Values are mean±SD.

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.
Figure 4. CD8+ (cluster of differentation) T-cell depletion reduced retinal vascular disease in oxygen-induced retinopathy (OIR). A, Representative flat mounts of retina from C57BL/6J mice at postnatal day (P) 18 with OIR stained with FITC (fluorescein isothiocyanate)-conjugated isolectin B4 to show the vasculature (green). OIR mice were administered the isotype control antibody IgG2a, a CD4-depletion antibody, or a CD8α-depletion antibody at P10. Top, The entire retina. Yellow boxes are presented at a higher magnification in the bottom. Arrowheads show areas of neovascularization, and asterisks indicate vaso-obliteration. Scale bar, 0.125 mm. B, Retinal neovascularization in OIR mice. n=9 mice per group from 3 independent experiments. Vascular leakage (C) and VEGF (vascular endothelial growth factor) protein levels (D) in retina of OIR mice and room air controls. n=5 to 6 mice per group. P values were calculated using a parametric 1-way ANOVA followed by the Holm-Sidak multiple comparisons test (B–D). Values are mean±SD.
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.
Figure 5. CD8+ (cluster of differentation) T cells have increased effector and memory functions in the retina of mice with oxygen-induced retinopathy (OIR). A and B, Representative flat mounts of retina at postnatal day 16 from CD8gfp+/− mice stained with tetramethylrhodamine-isolectin B4 to show the vasculature (red). Arrowheads indicate CD8+ T cells (green). Scale bar, 100 µm. A, CD8+ T cells were rare in the retina of room air control mice. B, In OIR, CD8+ T cells were frequently found near areas of retinal neovascularization. C, Quantitation of CD8+ T cells/field of retina from CD8gfp mice. **P<0.01 (Student t test). n=5 to 9 mice per group from at least 2 independent experiments. D, Representative flow cytometry dot plots of CD45hiCD3+ T cells were further gated to show CD4+ and CD8+ T cells and their effector and memory phenotypes in the retina of room air control and OIR C57BL/6J mice. The number of naive (E; CD44CD62L+), effector (F; CD44+CD62L), and memory (G; CD44+CD62L+) CD8+ T-cell subsets in room air control and OIR retina. n=3 to 4 mice per group. P values were calculated by the nonparametric Mann-Whitney U test (E–G). Values are mean±SD (D) and median with a 95% CI (E–G).

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).
Figure 6. CD8+ (cluster of differentation) T cells lacking IFNγ (interferon gamma), TNF (tumor necrosis factor), perforin, and granzymes are less able to cause retinal vascular disease in oxygen-induced retinopathy (OIR). Studies were performed in Rag1−/− (recombinant-activating gene 1 deficient) mice with OIR administered phosphate-buffered saline (PBS controls [Con]) or CD8+ T cells by adoptive transfer (AT) at postnatal day (P) 7 and P12. A, Representative flat mounts of retina at P18 stained with FITC (fluorescein isothiocyanate)-conjugated isolectin B4 to show the vasculature (green). Top, The entire retina of PBS control mice and mice adoptively transferred with WT (wild type) CD8+ T cells and CD8+ T cells deficient in IFNγ, TNF, Prf (perforin), or GzmA/B (granzymes A and B). Yellow boxes in the top are presented at higher magnification in the bottom. Arrowheads indicate areas of neovascularization. Asterisks denote vaso-obliteration. Scale bar, 0.125 mm. The adoptive transfer of WT CD8+ T cells augmented (B) neovascularization, (C) VEGF (vascular endothelial growth factor) protein levels, and (D) vascular leakage in the retina compared with OIR+WT CD8+ T cells. The adoptive transfer of CD8+ T cells deficient in IFNγ, TNF, Prf, or GzmA/B reduced (B) neovascularization and (C) VEGF protein levels. Only TNF−/− CD8+ T cells reduced (D) vascular leakage in the retina. n=10 to 12 mice per group from 3 independent experiments for neovascularization. n=6 to 8 mice per group for vascular leakage and VEGF protein levels. P values were calculated using a 1-way ANOVA followed by the Holm-Sidak multiple comparisons test (B and C) or the Kruskal-Wallis test (D). Values are mean±SD (B and C) and median with a 95% CI (D).

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).
Figure 7. CD8+ (cluster of differentation) T cells migrate into the retina via CXCR3 (C-X-C motif chemokine receptor 3) and CXCL10/IP-10 (interferon γ-induced protein 10) in phase II of oxygen-induced retinopathy (OIR). A, Chemokine and chemotactic cytokine protein levels measured in the retina of control and OIR C57BL/6J mice at postnatal day 13 using LEGENDplex. n=6 to 7 mice per group. Flow cytometry dot plots of CXCR3+CD8+ T cells in the blood of (B) room air control and (C) OIR mice at postnatal day 13. D, The number of circulating CXCR3+CD8+ T cells in mice with OIR compared with room air controls. n=10 to 11 mice per group from 3 independent experiments. Flow cytometry dot plots of CXCR3+CD8+ T cells in retina from C57BL/6J (E) control and (F) OIR mice at postnatal day 16. G, The number of CXCR3+CD8+ T cells in the retina of mice with OIR compared with room air controls. n=3 to 4 mice per group. P values were calculated using a parametric 2-way ANOVA followed by the Holm-Sidak multiple comparisons test (A), an unpaired t test with Welch correction (D), and the nonparametric Mann-Whitney U test (G). Values are mean±SD (A and D) and median with a 95% CI (G).
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.
Figure 8. Inhibition of CXCR3 (C-X-C motif chemokine receptor 3) reduced CD8+ T (cluster of differentation) cells and vascular disease in the retina of mice with oxygen-induced retinopathy (OIR). C57BL/6J mice with OIR were administered at postnatal day (P) 10 an anti-CXCR3 antibody or IgG isotype control antibody and retina examined at P16 and P18. A and B, Flow cytometry dot plots of CD8+ and CD4+ T cells in the retina of (A) OIR+anti-CXCR3 antibody and (B) OIR+IgG control antibody at P16. The number of (C) CD8+ T cells and (D) CD4+ T cells in the OIR retina following treatment with the CXCR3 blocking antibody compared with the control antibody. n=6 mice per group. E and F, Representative retinal flat mounts at P18 stained with tetramethylrhodamine-isolectin B4 to show the vasculature (red) from CD8gfp mice administered the (E) IgG control antibody or (F) anti-CXCR3 antibody. Arrows indicate CD8+ T cells (green). Scale bar, 150 µm. G, Quantitation of CD8+ T cells/field of retina from CD8gfp mice with OIR at P16. n=5 to 6 mice per group. H, Representative flat mounts of retina from mice with OIR at P18 stained with FITC (fluorescein isothiocyanate)-conjugated isolectin B4 to show the vasculature (green). Yellow boxes in the top are presented at a higher magnification in the bottom. Arrowheads indicate areas of neovascularization. Asterisks denote vaso-obliteration. Scale bar, 0.125 mm. CXCR3 inhibition reduced (I) neovascularization (n=9–10 mice per group from 3 independent experiments), (J) vascular leakage, and (K) VEGF (vascular endothelial growth factor) protein levels (n=5–6 mice per group) in retina of OIR mice. P values were calculated by the parametric unpaired t test with Welch correction. Values are mean±SD.

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

File (atvb_atvb-2022-318079_supp1.pdf)

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Go to Arteriosclerosis, Thrombosis, and Vascular Biology
Go to Arteriosclerosis, Thrombosis, and Vascular Biology
Arteriosclerosis, Thrombosis, and Vascular Biology
Pages: 522 - 536
PubMed: 36794587

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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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Keywords

  1. flow cytometry
  2. granzymes
  3. mice
  4. perforin
  5. retinal neovascularization

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Devy Deliyanti
Department of Anatomy and Physiology, School of Biomedical Sciences (D.D., J.L.W.-B.), University of Melbourne, Parkville, Victoria, Australia.
Department of Diabetes, Monash University, Melbourne, Victoria, Australia (D.D., J.L.W.-B.).
Department of Microbiology and Immunology, School of Biomedical Sciences, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Parkville, Victoria, Australia (W.A.F.).
Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia (W.A.F., T.G.).
Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia (W.A.F., T.G.).
Joseph A. Trapani
Sir Peter MacCallum Department of Oncology (J.A.T.), University of Melbourne, Parkville, Victoria, Australia.
Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia (J.A.T.).
Fabienne Mackay
QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia (F.M.).
Department of Anatomy and Physiology, School of Biomedical Sciences (D.D., J.L.W.-B.), University of Melbourne, Parkville, Victoria, Australia.
Department of Diabetes, Monash University, Melbourne, Victoria, Australia (D.D., J.L.W.-B.).

Notes

For Sources of Funding and Disclosures, see page 535.
Supplemental Material is available at Supplemental Material.
Correspondence to: Jennifer L. Wilkinson-Berka, PhD, Department of Anatomy and Neuroscience, University of Melbourne, Medical Bldg 181, Grattan St, Parkville, Victoria 3010, Australia. Email [email protected]

Disclosures

Disclosures None.

Sources of Funding

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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