Immune Amplification of Murine CD8+ Suppressor T Cells Induced via An Immune-Privileged Site: Quantifying Suppressor T Cells Functionally

BACKGROUND: CD8(+) suppressor T cells exert antigen-specific suppression of the expression of hypersensitivity by activated T cells. Therefore, CD8(+) suppressor T cells serve a major regulatory role for the control of active immunity. Accordingly, the number and/or activity of CD8(+) suppressor T cells should be influenced by an immune response to the antigen. To test this hypothesis we used an adoptive transfer assay that measures the suppression of the expression of delayed-type hypersensitivity (DTH) by CD8(+) suppressor T cells to quantify the antigen-specific suppression of DTH by these suppressor T cells. METHODS: Suppressor T cells were induced in the spleens of mice by the injection of antigen into the anterior chamber of an eye. Following this injection, the mice were immunized by the same antigen injected into the anterior chamber. Spleen cells recovered from these mice (AC-SPL cells) were titrated in an adoptive transfer assay to determine the number of AC-SPL cells required to effect a 50% reduction of antigen-induced swelling (Sw50) in the footpad of immunized mice challenged by antigen. RESULTS: Suppression of the expression of DTH is proportional to the number of AC-SPL cells injected into the site challenged by antigen. The number of AC-SPL cells required for a 50% reduction in DTH-induced swelling is reduced by injecting a cell population enriched for CD8(+) AC-SPL cells. Immunizing the mice receiving intracameral antigen to the same antigen decreases the RSw50 of AC-SPL cells required to inhibit the expression of DTH. CONCLUSIONS: The results provide the first quantitative demonstration that the numbers of antigen-specific splenic CD8(+) suppressor T cells are specifically amplified by antigen during an immune response

Molecular genetic screening and association studies of adult-onset primary open angle and congenital glaucomas

Glaucoma is a group of disorders with a broad range of clinical and histopathological manifestations. This condition presents itself in many different ways and with distinct shared characteristics that include optic neuropathy due to optic nerve head damage (cupping) and visual field dysfunction with or without presence of increased intraocular pressure (lOP). Glaucoma is the leading cause of irreversible blindness, which afflicts nearly 67 million people worldwide. It is considered the second most frequent cause of bilateral blindness, affecting 6.7 million people worldwide. By 2020, glaucoma is anticipated to affect 79.6 million people, increasing the number of bilaterally blind individuals due to glaucoma to 11.1 million. This thesis aimed to provide new insights into the molecular genetics of adult-onset Primary Open Angle Glaucoma (POAG) and a paediatric form of Primary Congenital Glaucoma (PCG). In the first portion of this work, the GLC1B locus on chromosome 2pl1.2-qI2.2 was investigated using extensive linkage and saturation mapping in order to reduce the region and to select potential candidate genes for screening 9 previously linked families. Genomic Convergence and Proteomic Streamlining methods were used to select and prioritise the most likely candidate genes. The prioritisation was based on tissue expression, bioinformatics, microarray data, as well as assessment of their protein- encoded functions. This investigation leads to the hypothesis that there may actually be two areas of interest for the GLCl B locus, one on the p arm and the other on the q arm of chromosome 2. Since at least three other independent studies shared a common overlapping area on the q arm of chromosome 2, single nucleotide polymorphisms (SNPs) from each of the genes within this common region were selected for a regional gene-specific association study. After genotyping our linked-GLC1B POAG families, sporadic cases and other matched control subjects, the genes that showed strong association were selected and screened for mutations. After direct sequencing of these genes, 62 DNA alterations (known and novel intronic and exonic variants) were observed and their disease-causing nature was carefully investigated. In conclusion, the GLC1B locus has been significantly reduced to a region of approximately 6.66-Mb. A total of 10 candidate genes were selected, individually sequenced and ultimately excluded as being involved in the aetiology of a group of GLC1B-linked POAG families. Further work is necessary to identify the defective gene(s) at the GLC1B locus. In the second portion of this thesis, the GLC3C locus on chromosome 14q24.3- q31.1, the study aimed to prioritise genes using similar methods to the strategies previously used to identify candidate genes within the GLC1B locus. These included Genomic Convergence, Proteomic Streamlining and the information from other linked families within the region. A total of 7 regional candidate genes were selected, sequenced and excluded as being involved in the aetiology of our GLC3C-linked family. Altogether, 80 DNA alterations (known and novel intronic and exonic variants) were observed in the sequenced genes and their disease-causing nature was carefully investigated. In addition, to the screening of the above-mentioned GLC3C candidate genes, a newly described PCG-causing gene, LTBP2, was fully sequenced and excluded in our GLC3C-linked family. Additionally, a total of 94 familial and sporadic PCG and 96 matched-control subjects were fully sequenced for the entire 36-coding exons of the LTBP2 gene. Only two heterozygous DNA alterations were identified in 2 PCG individuals, which were absent in 96 normal control subjects. Since no homozygous mutations were observed, further investigation is required to determine the disease- causing nature of these two DNA alterations. Therefore, this study was unable to replicate previously described mutations in the PCG subjects from Pakistan and Iran. Likewise, at least one other study of American PCG cases did not find any gene mutations in the LTBP2 gene, thus further confirming the observation of this study. As this gene is implicated in diseases with multiple clinical presentations, further investigation is necessary to determine the overall role that LTBP2 plays in the isolated PCG phenotype as well as in identifying the causative gene(s) within the GLC3C locus.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Role of Senescent Cells in Cutaneous Wound Healing

Cellular senescence has gained increasing attention in the field of aging research. Senescent cells have been implicated in biological aging processes, tumorigenesis, development, and wound repair amongst other processes and pathologies. Recent findings reveal that senescent cells can both promote and inhibit cutaneous wound healing processes. Relating senescent cells in acute and chronic wounds will help to clarify their role in wound healing processes and inform our understanding of senescent cell heterogeneity. To clarify this apparent contradiction and guide future research and therapeutic development, we will review the rapidly growing field of cellular senescence and its role in wound healing biology

The Induction of Circulating, ACAID-Inducing Monocytes Requires CCR2/CCL2-Dependent Migration of Circulating F4/80+ Cells into the Anterior Chamber

To determine the origin of peripheral blood mononuclear cells (PBMC) that activate regulatory T cells in anterior chamber-associated immune deviation (ACAID), fluorescein-labeled PBMC were intravenously injected into mice before the mice received an intracameral injection of antigen. Six-24 hr after intracameral injection, fluorescein-labeled PBMC increased in the iris. Twenty-four-48 hr labeled cells decreased in the iris and increased in the thymus and spleen. The entry of the labeled PBMC into the anterior chamber and subsequent production of PBMC that transfer ACAID required the expression of CCR2 by the PBMC and the production of the chemokine CCL2 by the recipient of the PBMC. The results suggest that the intracameral injection of antigen induces i) the infiltration of F4/80 + PBMC into the AC, ii) where these PBMC are converted to a regulatory phenotype, and iii) recirculate to activate T cells that suppress cell-mediated immunity

An intracameral injection of antigen induces in situ chemokines and cytokines required for the generation of circulating immunoregulatory monocytes.

Anterior Chamber-Associated Immune Deviation (ACAID) induced by an intracameral injection of antigen generates antigen-specific regulatory splenic T cells that suppress specifically cell-mediated immunity specific for the injected antigen. Circulating F4/80(+) cells recovered from mice receiving an intracameral injection of antigen are thought to be ocular in origin and induce the development of thymic and splenic regulatory T cells. We have shown previously that after the intracameral injection of antigen there is a CCR2/CCL2-dependent infiltration of circulating F4/80(+) cells into the anterior chamber associated with the generation of circulating, ACAID-inducing F4/80(+) monocytes. Here we tested the hypothesis that the intracameral injection of antigen induces events in the anterior chamber that are associated with the induction of circulating immunoregulatory monocytes that induce the suppression of cell-mediated immunity. The intracameral injection of antigen resulted in aqueous humor (i) a time- dependent increase of CCL2 and CCL7, (ii) a transient increase in TNF-α, and (iii) an infiltration of CD11b(hi), Gr1(hi) and F4/80(+) as well as F4/80(-) and Gr1(hi) peripheral blood cells into the anterior chamber. Further characterization of these F4/80(+) cells revealed that they are Ly 6C(hi), LY6G(lo) or negative, 7/4 (LY6B)(hi), CD115(+), CD45(+), CD49B(+), and CD62 L(+). Antibody-mediated neutralization of TGF-β in situ in the anterior chamber prevented the induction of circulating, ACAID-inducing monocytes and ACAID. These cells did not increase in the irides of ACAID-refractory CCR2-/- and CCL2-/- mice that received an intracameral injection of antigen. Our results extend our suggestion that ACAID is initiated as the result of a mild proinflammatory response to intracameral injection that results in the infiltration of a CCR2(+) subset of monocytes into the anterior chamber where there is a TGF-β-dependent induction of an immunosuppressive phenotype in the infiltrated monocytes that recirculate to induce antigen-specific regulatory T cells

The expansion of AC-SPL suppressor cells that suppress the expression of DTH is strain and antigen dose -dependent.

<p>Seven days after mice were immunized with TNP-BSA, a footpad was challenged with epicutaneous PCl. Footpad thickness was measured before and 24 hr after challenge. Five thousand AC-SPL cells were injected into a footpad of mice immunized with TNP-BSA immediately after the footpad was challenged with epicutaneous PCl. Footpad thickness was measured before and 24 hr after challenge and swelling computed. (A): Effect of immunizing dose of antigen on DTH-induced swelling in BALB/c (A) C57BL/6 mice (C), Generation of suppressive AC-SPL cells is antigen dose dependent (B): BALB/c, D: C57BL/6. Data represents the mean swelling +/− S.E.M. of 12 mice/group , 3 experiments. * p<0.05,NS: not significant.</p

Immunization reduces the RSw50 of AC-SPL cells.

<p>(A) BALB/c mice received an injection of TNP-BSA into the anterior chamber. Seven days after receiving an intracameral injection of TNP-BSA, some mice were immunized with TNP-BSA/CFA. Spleen cells were recovered from the immunized, AC-injected mice (AC-IMM) and mice that received an injection of antigen into the AC only (AC-ONLY) one week after the immunization of AC-injected mice or one week after intracameral injection only. Twenty-five thousand AC-SPL cells were injected into the footpads of TNP-BSA-immunized mice immediately after the footpads were challenged with epicutaneous PCl. (B) Immunization-induced increase in suppression is antigen-specific. Seven days after mice received an intracameral injection of TNP-BSA, the mice were immunized with TNP (AC-TNP), TNP-IMM or OVA (AC-TNP-OVA-IMM). Seven days after immunization, spleens were recovered from the mice and recovered spleen cells injected into the footpad of TNP-BSA-immunized mice immediately after the footpad was challenge with epicutaneous PCl. Swelling was measured 24 hr later. The data represents the mean RsW50 +/− the standard error of the mean for three experiments, 9 mice/group. *p<0.02.</p

The RsW50 of regulatory spleen cells is decreased by enriching for CD8⁺ regulatory spleen cells.

<p>The footpads of mice immunized with TNP-BSA and CFA 9 days previously received id CD8⁺ cells or unseparated AC-SPL cells prepared as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022496#pone-0022496-g001" target="_blank">Fig. 1</a> immediately after the footpad was challenged with epicutaneous PCl. Swelling was measured 24 hr later. The data is pooled from two separate experiments with six mice /group. NAÏVE: non-immunized mice, IMM: immunized mice that did not receive regulatory spleen cells, CD8⁺: immunized mice that received CD8⁺ regulatory spleen cells. p<0.01. Straight lines were generated by curve fit software.</p