Differentiation of endothelial cells from human umbilical cord blood AC133−CD14+ cells

Endothelial progenitor cells (EPCs) participate in neovascularization and are consistent with postnatal vasculogenesis. In vitro, they differentiate into endothelial cells (ECs). Prior reports have suggested that circulating human AC133(+) cells have the capacity to differentiate into ECs as progenitor cells. However, recent studies have demonstrated that circulating CD34(-)CD14(+) cells also have EPC-like properties in vitro and in vivo. We tested whether AC133(-)CD14(+) cells from human umbilical cord blood (HUCB) have the potential to differentiate into ECs. The AC133(-)CD14(+) cells were isolated from HUCB by magnetic bead selection and cultured on fibronectin-coated six-well trays in M199 medium supplemented with fetal bovine serum (FBS), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and insulin growth factor (IGF-1). The AC133(-)CD14(+) cells adhered slightly within 1 day of culture and subsequently underwent a distinct process of morphological transformation to spindle-shaped cells that sprouted from the edge of the cell clusters. After 14 days, the cells formed cord- and tubular-like structures. The AC133(-)CD14(+) cells showed a strong increase in the endothelial marker P1H12 over time, whereas CD14 decreased, and CD45 did not change, respectively. In addition, the cells expressed endothelial markers von Willebrand's factor (vWF), platelet/endothelial cell adhesion molecule-1 (PECAM-1), vascular endothelial growth factor receptor-1 (VEGFR-1)/Flt-1, VEGFR-2/Flk-1, eNOS, and VE-cadherin, but did not express Tie-2 after 7 days of culture. The present data indicate that AC133(-)CD14(+) cells from HUCB are able to develop endothelial phenotype with expression of endothelial-specific surface markers and even form cord- and tubular-like structures in vitro as progenitor cells.     

Characterization of an acute T lymphoblastic leukemia expressing the γ/δ T-cell receptor

Summary Leukemic cells of a 20 year old patient, suffering from acute lymphoblastic leukemia, were characterized by surface marker and functional analysis. A significant cell population within this type of leukemia expresses concomitantly the CD4 and CD8 antigen on the same cell and might represent a new differentiation stage of T-cells with the / receptor. The leukemic cells show a distinct pattern of growth response to mitogens and lymphokines, which might correlate to their differentiation stage. Moreover, a natural killer-like activity can be induced in these cells by IL-2.Abbreviations FITC fluorescein isothiocyanate - PE phycoerythrin - IL-2 interleukin 2; - / TCR gamma/delta T cell receptor - NK natural killer - PBL peripheral blood lymphocytes - T-ALL acute T lymphoblastic leukemia - ConA concanavalin A - PMA phorbol myristate acetate - BM bone marrow - IL-2R IL-2 receptor - TdT terminal deoxynucleotidyl transferase Supported by the Deutsche Forschungsgemeinschaft (DFG Wi-728/3-1)     

Engagement of NKp30 on Vδ1 T cells induces the production of CCL3, CCL4, and CCL5 and suppresses HIV-1 replication

Natural cytotoxicity receptors (NCRs) were originally identified as specific natural killer cell activating receptors that, on binding to their endogenous ligands, trigger the killing of tumor cell targets. We recently described the differentiation of a novel subset of NCR(+) Vδ1 T cells characterized by a remarkably high cytolytic potential against cancer cells. Here we demonstrate that the engagement of NKp30, one of the NCRs expressed de novo on Vδ1 T cells after stimulation, triggers the production of high levels of CCL3/MIP-1α, CCL4/ MIP-1β, and CCL5/RANTES but not of CXCL12/SDF-1. In turn, this NKp30-induced secretion of cc-chemokines is able to significantly suppress the replication of a CCR5 tropic strain of HIV-1 in CD4(+)/CCR5(+) infected PM1 cell lines. This experimental evidence disclosing an unanticipated antiviral function of NCR(+) Vδ1 T cells opens new avenues for understanding the pathogenic role and for manipulating the function of γδ T cells in HIV-1 infection.     

Effector Vγ9Vδ2 T cells dominate the human fetal γδ T-cell repertoire

γδ T cells are unconventional T cells recognizing antigens via their γδ T-cell receptor (TCR) in a way that is fundamentally different from conventional αβ T cells. γδ T cells usually are divided into subsets according the type of Vγ and/or Vδ chain they express in their TCR. T cells expressing the TCR containing the γ-chain variable region 9 and the δ-chain variable region 2 (Vγ9Vδ2 T cells) are the predominant γδ T-cell subset in human adult peripheral blood. The current thought is that this predominance is the result of the postnatal expansion of cells expressing particular complementary-determining region 3 (CDR3) in response to encounters with microbes, especially those generating phosphoantigens derived from the 2-C-methyl-d-erythritol 4-phosphate pathway of isoprenoid synthesis. However, here we show that, rather than requiring postnatal microbial exposure, Vγ9Vδ2 T cells are the predominant blood subset in the second-trimester fetus, whereas Vδ1⁺ and Vδ3⁺ γδ T cells are present only at low frequencies at this gestational time. Fetal blood Vγ9Vδ2 T cells are phosphoantigen responsive and display very limited diversity in the CDR3 of the Vγ9 chain gene, where a germline-encoded sequence accounts for >50% of all sequences, in association with a prototypic CDR3δ2. Furthermore, these fetal blood Vγ9Vδ2 T cells are functionally preprogrammed (e.g., IFN-γ and granzymes-A/K), with properties of rapidly activatable innatelike T cells. Thus, enrichment for phosphoantigen-responsive effector T cells has occurred within the fetus before postnatal microbial exposure. These various characteristics have been linked in the mouse to the action of selecting elements and would establish a much stronger parallel between human and murine γδ T cells than is usually articulated.Like conventional αβ T cells and B cells, γδ T cells use V(D)J gene rearrangement with the potential to generate a set of highly diverse receptors to recognize antigens. This diversity is generated mainly in the complementary-determining region 3 (CDR3) of the T-cell antigen receptor (TCR) or B-cell antigen receptor (1–3). The tripartite subdivision of lymphocytes possessing rearranged receptors into B cells, αβ T cells, and γδ T cells has been conserved since the emergence of jawed vertebrates more than 450 Mya (1). Recently, a similar division of variable lymphocyte receptor A (VLRA)⁺, VLRB⁺, and VLRC⁺ cells, resembling αβ T cells, B cells, and γδ T cells, respectively, has been found in jawless vertebrates (e.g., lamprey), showing the same basic principle of lymphocyte differentiation along two distinct T-cell–like lineages and one B-cell–like lineage (4). These evolutionary data highlight the importance of both γδ T cells and αβ T cells. A major difference between αβ T cells and γδ T cells is the way they recognize antigens. In contrast to conventional αβ T cells, γδ T cells are not dependent on classical MHC molecules presenting peptides. Based on the ligands that have been identified, it appears that some γδ TCRs can recognize antigens in an antibody-like fashion, whereas the TCRs of other γδ T-cell subsets can bind to nonclassical MHC-I or MHC-like proteins (2, 5–11). Although there are common characteristics among γδ T cells, some of which are shared with VLRC⁺ cells (4), it is clear that γδ T cells do not represent a homogenous population of cells with a single physiological role (12). γδ T cells expressing the TCR containing the γ-chain variable region 9 and the δ-chain variable region 2 (Vγ9Vδ2 T cells) are activated by microbe- and host-derived phosphorylated prenyl metabolites (phosphorylated antigens or “phosphoantigens”) derived from the isoprenoid metabolic pathway, the most active of which are microbial (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), produced by the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, and host isopentenyl pyrophosphate (IPP) (13). These phosphoantigens recently have been shown to be presented or to be sensed by the butyrophilin BTN3A1 (14–16). Although phosphoantigen-reactive Vγ9Vδ2 T cells were thought to be restricted to primates, there is recent evidence that Vγ9, Vδ2, and BTN3A1 genes are coconserved across a variety of placental mammals including primates, alpaca, armadillo, sloth, dolphin, dromedary, and orca, but not rodents (17). The recognition of phosphoantigens allows Vγ9Vδ2 T cells to develop potent antimicrobial immune responses or to promote the killing of transformed host cells that up-regulate IPP production (18, 19). Also, treatment of cells with the aminobisphosphonate family of drugs, of which zoledronate (Zometa) is the most potent member, leads to endogenous IPP accumulation (18). This feature has been used to develop clinical trials targeting Vγ9Vδ2 T cells of patients with leukemia or solid cancers (19, 20). Vγ9Vδ2 T cells represent the main population of γδ T cells in adult human peripheral blood: About 50–90% of γδ T cells in the circulation express this combination of Vγ and Vδ chains because of postnatal expansion (21). In contrast, γδ T cells expressing the Vδ1 chain, which can pair with a variety of Vγ chains, are enriched in adult tissues such as the gut (21).Instead of being regarded as just an immature version of the adult immune system, the immune system in early life increasingly is being recognized as different, with a bias toward the induction of a Th2 response or of immune tolerance (22–26). Indeed, one of the last cytokines to reach adult levels after birth is the Th1-promoting cytokine IL-12 (IL-12p70) (27). Originally proposed as a hypothesis by Adrian Hayday (1), there is increasing evidence, including our own results, that γδ T cells are important in early life (28–33). Although in humans circulating T cells can be detected as early as 12.5 wk gestation, most information on T cells in early life, including γδ T cells, is derived from studies on cord blood at term delivery (>37 wk gestation) (34). We hypothesized that the human fetus could produce particular fetal type of γδ T cells, as has been well established in the mouse model (35–37). Furthermore, it has been reported recently that fetal and adult hematopoietic stem cells can give rise to distinct T-cell lineages in humans, with a bias toward immune tolerance in the fetus (24).Here we found that, unexpectedly, fetal blood around midgestation (before 30 wk) contained high levels of Vγ9Vδ2 T cells. These lymphocytes expressed a semi-invariant TCR, were phosphoantigen reactive, and showed a preprogrammed effector potential, suggesting that these γδ T cells may fulfill an important role in the immunosurveillance of fetal tissues.     

Vβ2 natural killer T cell antigen receptor-mediated recognition of CD1d-glycolipid antigen

Natural killer T cell antigen receptors (NKT TCRs) recognize lipid-based antigens (Ags) presented by CD1d. Although the TCR α-chain is invariant, NKT TCR Vβ exhibits greater diversity, with one (Vβ11) and three (Vβ8, Vβ7, and Vβ2) Vβ chains in humans and mice, respectively. With the exception of the Vβ2 NKT TCR, NKT TCRs possess canonical tyrosine residues within complementarity determining region (CDR) 2β that are critical for CD1d binding. Thus, how Vβ2 NKT TCR docks with CD1d-Ag was unclear. Despite the absence of the CDR2β-encoded tyrosine residues, we show that the Vβ2 NKT TCR engaged CD1d-Ag in a similar manner and with a comparable affinity and energetic footprint to the manner observed for the Vβ8.2 and Vβ7 NKT TCRs. Accordingly, the germline-encoded regions of the TCR β-chain do not exclusively dictate the innate NKT TCR-CD1d-Ag docking mode. Nevertheless, clear fine specificity differences for the CD1d-Ag existed between the Vβ2 NKT TCR and the Vβ8.2 and Vβ7 NKT TCRs, with the Vβ2 NKT TCR exhibiting greater sensitivity to modifications to the glycolipid Ag. Furthermore, within the Vβ2 NKT TCR-CD1d-αGalCer complex, the CDR2β loop mediated fewer contacts with CD1d, whereas the CDR1β and CDR3β loops contacted CD1d to a much greater extent compared with most Vβ11, Vβ8.2, and Vβ7 NKT TCRs. Accordingly, there is a greater interplay between the germline- and nongermline-encoded loops within the TCR β-chain of the Vβ2 NKT TCR that enables CD1d-Ag ligation.     

Age-associated alteration of γδ T-cell repertoire and different profiles of activation-induced death of Vδ1 and Vδ2 T cells

It has been suggested that γδ T cells are involved in certain autoimmune disorders. To establish reference data for clinical studies to explore the role of γδ T cells in autoimmune bone marrow failure syndrome, we examined the γδ T-cell repertoire in 120 healthy Japanese individuals by flow cytometry. The average numbers of T lymphocytes in blood were as follows: 1,084 ± 369 (SD) αβ T cells, 68 ± 44 γδ T cells, 16 ± 12 Vδ1 T cells, and 43 ± 36 Vδ2 T cells (/μl). Absolute numbers of γδ T cells decreased with aging (R = -0.378, P < 0.001). The decrease of γδ T cells was the result of reduction of Vδ2, but not of Vδ1, T cells. Numbers of Vδ2 T cells were significantly higher in male than in female donors (P = 0.007). The Vδ2 T cells but not Vδ1 T cells showed a rapid reduction in cell numbers on mitogen stimulation, which was accompanied by modest down-regulation of Bcl-2 protein expression. These results indicate that age and gender have a major impact on γδ T-cell repertoire in Japanese donors, as well as European and American donors. The age-related decrease of Vδ2 T cells may be explained by their susceptibility to activation-induced cell death.     

Gastric cancer cells induce human CD4~+ Foxp3~+ regulatory T cells through the production of TGF-β1

AIM: To elucidate the molecular and cellular features responsible for the increase of regulatory T cells (Tregs) in gastric cancer. METHODS: The frequencies of CD4 + Foxp3 + Tregs and the level of transforming growth factor-β1 (TGF-β1) were analyzed from 56 patients with gastric cancer byflow cytometry and enzyme-linked immunosorbent assay respectively. Foxp3 gene expression was analyzed by real-time polymerase chain reaction. The gastric cancer microenvironment was modeled by establishing the coculture of ...     

Analysis of memory-like natural killer cells in human cytomegalovirus-infected children undergoing αβ+T and B cell-depleted hematopoietic stem cell transplantation for hematological malignancies

We analyzed the impact of human cytomegalovirus infection on the development of natural killer cells in 27 pediatric patients affected by hematological malignancies, who had received a HLA-haploidentical hematopoietic stem cell transplantation, depleted of both α/β+ T cells and B cells. In line with previous studies in adult recipients of umbilical cord blood transplantation, we found that human cytomegalovirus reactivation accelerated the emergence of mature natural killer cells. Thus, most children displayed a progressive expansion of a memory-like natural killer cell subset expressing NKG2C, a putative receptor for human cytomegalovirus, and CD57, a marker of terminal natural killer cell differentiation. NKG2C⁺CD57⁺ natural killer cells were detectable by month 3 following hematopoietic stem cell transplantation and expanded until at least month 12. These cells were characterized by high killer Ig-like receptors (KIRs) and leukocyte inhibitory receptor 1 (LIR-1) and low Siglec-7, NKG2A and Interleukin-18Rα expression, killed tumor targets and responded to cells expressing HLA-E (a NKG2C ligand). In addition, they were poor Interferon-γ producers in response to Interleukin-12 and Interleukin-18. The impaired response to these cytokines, together with their highly differentiated profile, may reflect their skewing toward an adaptive condition specialized in controlling human cytomegalovirus. In conclusion, in pediatric patients receiving a type of allograft different from umbilical cord blood transplantation, human cytomegalovirus also induced memory-like natural killer cells, possibly contributing to controlling infections and reinforcing anti-leukemia effects.     

Up-regulation of cytolytic functions of human Vδ2-γ T lymphocytes through engagement of ILT2 expressed by tumor target cells

In humans, the majority of peripheral blood γδ T cells expresses Vγ9Vδ2 T-cell receptors (TCR) and recognize nonpeptidic phosphorylated antigens. In contrast, most tissue-derived γδ T cells, which are located mainly in spleen and epithelia, preferentially use Vδ1 or Vδ3 chains paired with diverse Vγ chains to form their TCR. Our knowledge about the antigenic specificity and costimulation requirements of human Vδ2(-) γδ T cells remains limited. In an attempt to address this important issue, we characterized the specificity of a monoclonal antibody (mAb 256), screened for its ability to specifically inhibit cytolytic responses of several human Vδ2(-) γδ T-cell clones against transformed B cells. We show that mAb 256 does not target a TCR ligand but blocks key interactions between non-TCR molecules on effector γδ T cells and ILT2 molecule, expressed by tumor targets. In line with the previously reported specificity of this NK receptor for classic and nonclassic major histocompatibility complex (MHC) class I molecules, blockade of MHC class I/ILT2 interactions using MHC class I- or ILT2-specific mAbs and ILT2-Fc molecules inhibited tumor-induced activation of Vγ8Vδ3 T-cell clones. Therefore, this study describes a new cytotoxic T lymphocyte activation pathway involving MHC class I engagement on γδ T cells.     

DC-like cell-dependent activation of human natural killer cells by the bisphosphonate zoledronic acid is regulated by γδ T lymphocytes

Bisphosphonates are mainly used for the inhibition of osteoclast-mediated bone resorption but also have been shown to induce γδ T-cell activation. Using IL-2-primed cultures of CD56(+) peripheral blood mononuclear cells, we show here that zoledronic acid (zoledronate) could induce IFN-γ production not only in γδ T lymphocytes but, surprisingly, also in natural killer (NK) cells in a manner that depended on antigen-presenting cells, which share properties of inflammatory monocytes and dendritic cells (DCs; here referred to as DC-like cells). In the presence of γδ T lymphocytes, DC-like cells were rapidly eliminated, and NK cell IFN-γ production was silenced. Conversely, in the absence of γδ T lymphocytes, DC-like cells were spared, allowing NK cell IFN-γ production to proceed. γδ T cell-independent NK cell activation in response to zoledronate was because of downstream depletion of endogenous prenyl pyrophosphates and subsequent caspase-1 activation in DC-like cells, which then provide mature IL-18 and IL-1β for the activation of IL-2-primed NK cells. Pharmacologic inhibition of caspase-1 almost abolished IFN-γ production in NK cells and γδ T lymphocytes, indicating that caspase-1-mediated cytokine maturation is the crucial mechanism underlying innate lymphocyte activation in response to zoledronate.     

Do the human umbilical cord blood CD34+ progenitor cells home in the pancreas and kidney of diabetic mice?

Type 1 diabetes is a chronic metabolic disorder in which pancreatic islet β cells are irreversibly destroyed by autoimmunity. Many studies suggest great promise for the utility of human umbilical cord blood (HUCB) stem cells as a cure for diabetes; however, the mechanism for their effect requires further elucidation. This study investigated the presence of human DNA in the pancreas and kidney of diabetic mice treated with HUCB CD34+ cells. Eighteen albino male mice were equally and randomly divided into three groups: normal control group, diabetic untreated streptozotocin (STZ) group, and diabetic STZ-treated group. Diabetes was induced by intraperitoneal (IP) injection of STZ (180 mg/kg). CD34+ progenitor cells were separated from HUCB and injected intravenously in dose of 10⁶ cells/mouse. Blood glucose was measured every 2 days for 3 weeks. Mice were sacrificed after 3 weeks, and real-time PCR analysis was conducted for the presence of human-specific ALU sequence in the pancreata and kidneys from all animals. Injection of CD34+ cells caused significant improvement in blood glucose level (230?±?50 mg/dl in treated group vs. 590?±?24 mg/dl in untreated group, p?=?0.001). Real-time PCR analysis showed negative results in the control and untreated groups, while in the treated group, engraftment of the HUCB CD34 cells was positive in 100 % of the kidneys with a mean transplanted cell percentage 1.8?±?0.98 % and 66 % of the pancreata with a mean 0.41?±?0.42 %. A significant negative correlation was found between the concentration of the ALU sequence in the pancreata and the change of glucose level in the treated group (p value?=0.03 and r?=??0.6). HUCB CD34 cells engraft in the pancreas of the diabetic mice and improve hyperglycemia. Based on our results, HUCB CD34 cell transplantation may provide a potential therapy for human diabetes mellitus.     

Evaluation of TCR Vβ subfamily T cell expansion in NOD/SCID mice transplanted with human cord blood hematopoietic stem cells

Examination of the T cell receptor (TCR) gene repertoire is important in the analysis of the immune status of models, because clonal expansion of T cells permits the identification of specific antigen responses of T cells. Little is known about T-cell immunity in the humanized NOD/SCID mouse model. TCR Vβ repertoire usage and clonality were analyzed to investigate the distribution and clonal expansion of TCR Vβ subfamily T cells in NOD/SCID mice transplanted with human cord blood (CB) hematopoietic stem cells. The NOD/SCID mice were sublethally irradiated (⁶⁰Co, 300cGy) to eliminate residual innate immunity in the host. The experimental mice were transplanted intravenously with CB CD34⁺ cells sorted by MACS. After 6 weeks, RNA was obtained from peripheral blood, bone marrow and thymus of the study animals. The gene expression and clonality of the TCR Vβ repertoire were determined by RT-PCR and GeneScan techniques. A restricted range of TCR Vβ usage was exhibited in the bone marrow of mice, which included TCR Vβ 1, 2, 9, 13 and 19. Further, oligoclonal expression of some TCR Vβ subfamilies (Vβ9, 13, 19) was identified by GeneScan technique. To investigate the reason for oligoclonal expansion of the TCR Vβ subfamily T cells from CB in mouse models, the T-cell culture with tissue-antigen of NOD/SCID mouse was performed in vitro. The cells from peripheral blood mononuclear cells and bone marrow, spleen, thymus in NOD/SCID mice were frozen and thawed, and used as tissue-antigen. CB mononuclear cells were separately cultured with the component from those murine cells for 15–20 days. Oligoclonal expression or oligoclonal trend of some TCR Vβ subfamilies (Vβ10, 11 and Vβ2, 15, 16, 19) was detected in T cells after stimulation with tissue-antigen of NOD/SCID mouse. Interestingly, a similar clonal expansion of the TCR Vβ11 subfamily was found in T cells cultured with peripheral blood, bone marrow and spleen respectively. The TCR Vβ subfamily T cells could be reconstituted in humanized NOD/SCID mouse transplanted with CD34⁺ cells from CB. The restricted expression and clonal expansion of some CB T cell clones may be induced by tissue-antigens of NOD/SCID mice.     

The majority of the in vitro erythroid expansion potential resides in CD34? cells,outweighing the contribution of CD34+ cells and significantly increasing the erythroblast yield from peripheral blood samples

The study of human erythropoiesis in health and disease requires a robust culture system that consistently and reliably generates large numbers of immature erythroblasts that can be induced to differentiate synchronously. We describe a culture method modified from Leberbauer et al. (2005) and obtain a homogenous population of erythroblasts from peripheral blood mononuclear cells (PBMC) without prior purification of CD34⁺ cells. This pure population of immature erythroblasts can be expanded to obtain 4×10⁸ erythroblasts from 1×10⁸ PBMC after 13–14 days in culture. Upon synchronized differentiation, high levels of enucleation (80–90%) and low levels of cell death (<10%) are achieved. We compared the yield of erythroblasts obtained from PBMC, CD34⁺ cells or PBMC depleted of CD34⁺ cells and show that CD34⁻ cells represent the most significant early erythroid progenitor population. This culture system may be particularly useful for investigating the pathophysiology of anemic patients where only small blood volumes are available.     

Notch-Hes1 pathway is required for the development of IL-17-producing γδ T cells

Unlike conventional T cells, which are exported from the thymus as naive cells and acquire effector functions upon antigen encounter in the periphery, a subset of γδ T cells differentiates into effectors that produce IL-17 within the fetal thymus. We demonstrate here that intrathymic development of the naturally occurring IL-17-producing γδ T cells is independent of STAT3 and partly dependent on RORγt. Comparative gene-expression analysis identified Hes1, one of the basic helix-loop-helix proteins involved in Notch signaling, as a factor specifically expressed in IL-17-producing γδ T cells. Hes1 is critically involved in the development of IL-17-producing γδ T cells, as evidenced by their severe decrease in the thymi of Hes1-deficient fetal mice. Delta-like 4 (Dll4)-expressing stromal cells support the development of IL-17-producing γδ T cells in vitro. In addition, conditional Hes1 ablation in peripheral γδ T cells decreases their IL-17 production but not their IFN-γ production. These results reveal a unique differentiation pathway of IL-17-producing γδ T cells.     

The dominant human conjunctival epithelial CD8αβ+ T cell population is maintained with age but the number of CD4+ T cells increases

The conjunctiva is a highly specialized ocular mucosal surface that, like other mucosa, houses a number of leukocyte populations. These leukocytes have been implicated in age-related inflammatory diseases such as dry-eye, but their phenotypic characteristics remain largely undetermined. Existing literature provides rudimentary data from predominantly immunohistochemical analyses of tissue sections, prohibiting detailed and longitudinal examination of these cells in health and disease. Using recovered cells from ocular surface impression cytology and flow cytometry, we examined the frequency of leukocyte subsets in human conjunctival epithelium and how this alters with age. Of the total CD45+ leukocyte population within the conjunctival epithelium, 87% [32–99] (median) [range] comprised lymphocytes, with 69% [47–90] identified as CD3 + CD56- T cells. In contrast to peripheral blood, the dominant conjunctival epithelial population was TCRαβ + CD8αβ + (80% [37–100]) with only 10% [0-56%] CD4+ cells. Whilst a significant increase in the CD4+ population was seen with age (r = 0.5; p < 0.01) the CD8+ population remained unchanged, resulting in an increase in the CD4:CD8 ratio (r = 0.5;p < 0.01). IFNγ expression was detectable in 18% [14–48] of conjunctival CD4+ T cells and this was significantly higher among older individuals (<35 years, 7[4–39] vs. >65 years, 43[20–145]; p < 0.05). The elevation of CD4+ cells highlights a potentially important age-related alteration in the conjunctival intra-epithelial leukocyte population, which may account for the vulnerability of the aging ocular surface to disease.