CD1a-autoreactive T cells are a normal component of the human alpha-beta T cell repertoire

Source: de Jong A, Peña-Cruz V, Cheng TY, Clark RA, Van Rhijn I, Moody DB. CD1a-autoreactive T 

cells are a normal component of the human αβ T cell repertoire. Nat Immunol. 2010 Dec;11(12)

:1102-9. Epub 2010 Oct 31. PubMed PMID:21037579.

 

Members of CD1 system (CD1a, CD1b, CD1c and CD1d) bind and present lipid antigens from mammalian cells and bacteria. Based on results of several experimental studies, it is though that CD1 system is a system which allows T cells to survey CD1-expressing antigen presenting cells for changes in lipid content caused by infection, inflammation or malignancy. Studies suggest that human CD1a, CD1b and CD1c proteins and their non-human orthologs play important roles in immune response. Nearly all mammalian genomes have one or more genes encoding CD1a, and no mammalian species is known to have survived without genes encoding CD1 proteins. This retention of large gene families encoding CD1 proteins in most mammals suggests that each type of CD1 protein has a non-redundant and physiologically important role. Molecular and cellular evidences show that CD1a, CD1b, CD1c and CD1d differ in their transcriptional regulation, patterns of tissue expression, sub-cellular trafficking and antigen grove size. However, it is not known whether these differences also translate in to functional differences in the responding T cell population.

Human CD1-autoreactive T cells were discovered nearly two decades ago. Most of the insight into function of these cells has been obtained by several T cell clones rather than direct measurement of polyclonal T cells from blood and tissue. These studies have helped in assigning certain unchanging aspects of T cell phenotype such as TCR structure, expression of CD4 and CD8 co-receptors, antigens recognized and molecular mechanism of activation. However, long term culture of human T cells promotes growth of clones with in vitro growth advantage, which is not representative of in vivo repertoires. Even the best available reagents are limited in their ability to track fresh polyclonal T cells, especially CD1-autoreactive T cells. CD1-restricted T cells are able to recognize endogenous lipid antigens. The extent to which a lipid self antigen can be used to track larger pool of autoreactive T cells is unknown.

The authors sought to overcome these problems by designing a system in which cells provide a diverse pool of lipid antigens for loading onto CD1. The authors further wanted to minimize or remove the normally strong alloreactive responses to MHC class I and class II proteins that would confound with CD1-reactive responses. The authors thus transfected plasmids encoding human CD1 molecules into human myelogenous leukemia cells (K562 cells) that have low to absent expression of MHC. This resulted in MHCs that can be universally used with MHC-mismatched subjects. As these cells express a wide array of self lipid antigens, CD1 autoreacitivity can be detected broadly without prior knowledge of antigen structures. The authors observed that this method detected stronger and more frequent responses to CD1a than to any other human CD1 molecule. The authors also found that CD1a reactive T cells were fundamentally different from CD1d-reactive invariant NKT cells on the basis of their TCR patterns and effector functions. Another important observation that the authors made was that CD1a-autoreactive T cells expressed skin-homing markers and could be isolated from the skin and activated by epithelial CD1a-expressing Langerhans cells (LC). This study identified CD1a-autoreactive cells as subset of human T cells repertoire and defined CD1a as a target of the cells of Th22 helper T cell subset, which suggests a new model of LC-T cells interaction in epithelial homeostasis.

The results are described in detail below:

• CD1a-autoreactive T cells in peripheral blood: The investigators first developed a system that can measure the CD1 autoreactivity of non-clonal T cell population ex vivo. They transfected K562 cells, which have low surface density of MHC proteins, with genes encoding CD1a, CD1b, CD1c or CD1d. This resulted in high surface expression of these proteins. The investigators then confirmed the T cell-activating function of each transfected CD1 molecule by the presentation of known exogenous lipid antigens to CD1-restricted control T cell lines. For this experiment, IL-2 was analyzed in supernatant of K562 cells trasnfected with a particular CD1 molecule and incubated with T cell lines recognizing that particular CD1 molecule and exogenous lipid antigen specific for that CD1. For example, they used dideoxymycobactin (DDM) for CD1a, glucose monomycolate (GMM) for CD1b, mannosyl phosphomycoketide (MPM) for CD1c and alpha-galactosylceramide for CD1d. To determine whether CD1-restricted T cells can be detected in human peripheral blood, the investigators analyzed blood samples from 14 donors. Thus, the authors first stimulated polyclonal T cells with DCs expressing CD1a, CD1b, CD1c and CD1d and then analyzed IFN gamma secretion by T cells using CD1-trasnfected K562 cells as APCs. All the 14 donors responded to K562-CD1a cells and the mean responses were greater than those to CD1b, CD1c and CD1d. Three of the 14 donors responded to K562-CD1b, while only one of the 14 donors responded to CD1c or CD1d. Furthermore, the authors took five donors and expanded T cells in the presence of DCs and then tested the restriction of resulting short term T cell lines with K562-CD1a cells in the presence or absence of a CD1a blocking monoclonal antibody. They looked for IL-2 response in these cells. The results showed that a good response was observed when no antibody was present, however when blocking antibody was present , expression of Il-2 was reduced to background level. These results indicated that CD1a on the APCs was required for T cell response. The investigators then took a separate group of 14 donors, stimulated them with autologous DCs and analyzed clonal precursor frequency ex vivo. They generated a panel of 1291 T cell clones and the determined what percentage of autoreactive T cell clones secreted IL-2 in response to K562-CD1a cells relative to mock transfectants. They observed that one in every 50 T cell clones was CD1a autoreactive in 12 out of 14 samples. They analyzed co-receptor expression in 12 samples and found 11 CD4+ single positive clones, 1 CD8+ single positive clone, and no CD4- or CD8- clones. They next sequenced TCR alpha- and beta- chains and found that none of the CD1a-reactive clones expressed the invariant Valpha24 (TRAV10) or Vbeta11 (TRBV25), which are found on human NKT cells. The authors also observed that none of the clones expressed identical CDR3 sequences. Thus, they concluded that CD1a-autoreactive T cells are common in peripheral blood of humans and do not have the highly conserved TCR sequences seen in invariant NKT cells. The authors took eight random donors and purified CD45RO+ memory T cells, treated them with K562-CD1a cells and detected IFN-gamma response using ELISPOT assay. All the eight donors showed IFN-gamma response. Antibody blocking experiments showed that CD1a was necessary and sufficient for the response. The authors estimated the frequency of CD1a-autoreactive T cells to be between 0.02% and 0.4%. As not all CD1a-reactive T cells produce IFN-gamma, the authors performed ex vivo analysis of expanded cell population and found that T cell response to CD1a were of greater magnitude than those to other isoforms of CD1. Taken together these results indicate that CD1a-autoreactive T cells are abundant in blood without immune stimulation and were present in most or all donors. Thus, these cells can be considered to constitute a subset of the normal human alpha beta T cell repertoire.
  
• Homing of CD1a-autoreactive T cells to skin: In the periphery, CD1a is found predominantly in skin. Thus, the investigators hypothesize that CD1a-autoreactive T cells in the peripheral circulation might normally home to and localize in the skin. The investigators first determined whether CD1a-autoreactive T cells recovered from the blood expressed skin-homing markers. As it is known that surface expression of homing markers can be rapidly altered during in vitro culture, the authors sorted peripheral blood T cells ex vivo immediately after isolation on the basis of the expression of the membrane marker CD45RO and cutaneous lymphocyte antigen (CLA). The authors earlier made a chance finding of which they took advantage in these experiments. Donor 8 had an autoreactive T cell clone that was isolated from each of the three samples taken for the study. This indicated that these T cells had undergone clonal expansion in vivo. The authors confirmed the clonality of each isolate by expression of identical TCR alpha and TCR beta chains. They were than able to measure the presence of this CD1a specific clone in the naïve T cell fraction, memory T cell fraction and skin-homing memory T cell fraction of the peripheral blood. They found the PCR products for clonotypic primers almost exclusively in the memory T cell fraction (CD45RO+ CLA+). These results indicated that in donor 8, CD1a-autoreactive T cells were CLA+ memory T cells and that CD1a-autoreactive T cells can be a part of skin-homing T cell subset.
  
• CD1a-dependent IL-22 production: The investigators next stimulated CD1a-reactive T cell lines with K562-CD1a cells and measured cytokine mRNA by real time PCR. These cytokines included IFN-gamma, IL-2, those associated with T helper 1, T helper 2 and IL-17 producing subsets of MHC-restricted repertoire as well as cytokines produced by CD1d restricted NKT cells. The investigators compared the mRNA produced in response to K562-CD1a cells treated with control IgG with the mRNA produced in response to K562-CD1a cells treated with the CD1a-blocking mAb. Of the eight donors studies in this work, two showed an up-regulation of IFN-gamma alone while five showed an up-regulation of IL-22. The authors also detected secretion of IL-22 protein a dose dependent manner in response to CD1a. The authors observed a lack of substantial CD1a-dependent up-regulation of IL-17 mRNA in most donors and thus they considered that IL-22 and IL-17 are not coordinately upregulated in human CD1a-autoreactive T cells. The authors next performed intracellular cytokine staining of IL-17 and IL-22 in T cell lines from donors showing IL-22 upregulation. The experiments were performed without any stimulation and with stimulation with PMA and ionomycin. The results showed that even in the presence of stimulators, IL-17 production is not induced in IL-22 producing T cell lines. When the authors repeated same experiments in T cell fraction of total PBMCs, the presence of stimulus led to induction of production of both cytokines. These data support the idea that IL-17 and IL-22 represent the effector molecules produced by distinct T cell population in humans. These results also indicate that CD-1a-autoreactive T cells are the ones that produce IL-22. Thus, till now the authors found that a subset of CD1a-autoreactive T cell clones, selected on the basis of their production of IFN-gamma and confirmed for clonality by Vbeta PCR analysis, expressed either IFN-gamma alone or both IFN-gamma and IL-22 in various ratios in response to CD1a. These data provided direct evidence of dual cytokine-producing CD1a-autoreactive T cells at the clonal level and suggested that CD1a-dependent IFN-gamma responses observed by the authors in their previous study were probably both Th1 and Th22-Th1 cells.
  
• Skin-homing Th22 cells recognize CD1a: In addition to producing IL-22, Th22 cells are also characterized by expression of the aryl hydrocarbon receptor and expression of the chemokine receptors CCR6, CCR4 and CCR10, which promote skin homing. The authors observed that CD1a-autoreactive T cell lines expressed the aryl hydrocarbon receptor. The authors next sorted memory CD4+ T cells into the following fractions: CCR6+CXCR3+CCR4-CCR10-, CCR6+CXCR3-CCR4+CCR10- and CCR6+CXCR3-CCR4+CCR10+ and measured cytokine profiles in these fractions by RT-PCR. The results confirmed that these three populations were enriched in Th1, Th17 and Th22 cells, respectively. The authors then performed single in vitro expansion of sorted Th1 or Th22 T cell fractions with DCs and cultured them with K562-CD1a cells preincubated with mAb to CD1a or control IgG and observed that in vitro expansion caused twofold or greater CD1a-dependent up-regulation of cytokine mRNA in five of six donor. They also observed that Th22 cells showed the greatest and most frequent CD1a responses. IL-2 and IL-22 were the most dominantly upregulated cytokines in these cells. These results indicate that Cd1a-autoreactive T cells are found in human h22 subset and thus CD1a is a target for Th22 cells.
  
• CD1a-autoreactive responses from skin: To assess whether CD1a-autoreactive T cells are present in the skin, the investigators isolated T cells from human skin biopsy specimens and assessed CD1a recognition. During the initial screen, skin T cell clones were isolated whose activation was blocked by mAB to CD1a. The authors next assessed CD1a-dependent production of IL-22 in polyclonal T cells. In case of two samples (out of 3), lymphocytes isolated from human skin showed substantial production of IL-22 in response to K562-CD1a cells but not to K562-CD1b or K562-CD1c cells. These results indicated the dominance of CD1a-mediated responses in healthy human skin and their ability to produce IL-22.