Thymus Leukemia Antigen Research Articles

Innate CD8αα+ lymphocytes enhance anti-CD40 antibody-mediated colitis in mice.

IntroductionImmune responses in the intestines require tight regulation to avoid uncontrolled inflammation. We previously described an innate lymphocyte population in the intestinal epithelium (referred to as innate CD8αα+, or iCD8α cells) that can protect against gastrointestinal infections such as those mediated by Citrobacter rodentium.MethodsHere, we have evaluated the potential contribution of these cells to intestinal inflammation by analyzing inflammation development in mice with decreased numbers of iCD8α cells. We also determined the potential of iCD8α cells to secrete granzymes and their potential role during inflammatory processes.ResultsWe found that iCD8α cells play a pro‐inflammatory role in the development of disease in a colitis model induced by anti‐CD40 antibodies. We further found that the effects of iCD8α cells correlated with their capacity to secrete granzymes. We also observed that the pro‐inflammatory properties of iCD8α cells were controlled by interactions of CD8αα homodimers on these cells with the thymus leukemia antigen expressed by intestinal epithelial cells.ConclusionsOur findings suggest that iCD8α cells modulate inflammatory responses in the intestinal epithelium, and that dysregulation of iCD8α cells effector functions may enhance disease. We propose that one of the mechanism by which iCD8α cells enhance inflammation is by the secretion of granzymes, which may promote recruitment of infiltrating cells into the epithelium.

Dysregulation of iCD8α cells promotes susceptibility to intestinal inflammation

Abstract iCD8α cells are a population of innate immune cells present in the intestinal intraepithelial lymphocyte (IEL) compartment, characterized by the expression of CD8αα. We have shown that iCD8α cells play a critical role during immune responses in the intestinal mucosa, however, little is known about the role of iCD8α cells during inflammatory processes of the intestines. The thymus leukemia (TL) antigen is predominantly expressed by intestinal epithelial cells and preferentially interacts with CD8aa. We have shown that CD8αα+ IEL from TL-deficient mice have increased cytokine production and proliferation, indicating that TL controls CD8αα+ IEL effector functions. Rag-deficient mice facilitate the study of innate immune cells, and are an excellent model for the study of iCD8α cells because these cells are the only CD8αα+ IEL present in these mice. Here we present evidence showing that TL regulates the functions of iCD8α cells. Using the anti-CD40 antibody model of colitis, we show that TL−/−Rag-2−/− mice lose more weight, have higher mortality, have more colon damage, and present higher levels of pro-inflammatory cytokines (IFN-γ, IL-6 and OPN) than colons from Rag-2−/− mice. Interestingly, we show that colons and iCD8α cells from TL-deficient mice produce more granzyme than colons or iCD8α cells from TL-competent mice. Moreover, stimulation of iCD8α cells with the pro-inflammatory cytokine IL-12 in vitro induces granzyme secretion. Therefore, we propose that the increased colon inflammation observed in TL-deficient mice is due to: a) dysregulation of iCD8α cells by the lack of TL, and b) increased granzyme secretion by iCD8α cells which may serve as a pro-inflammatory signal.

CD8αα+ Innate-Type Lymphocytes in the Intestinal Epithelium Mediate Mucosal Immunity

Innate immune responses are critical for mucosal immunity. Here we describe an innate lymphocyte population, iCD8α cells, characterized by expression of CD8α homodimers. iCD8α cells exhibit innate functional characteristics such as the capacity to engulf and kill bacteria. Development of iCD8α cells depends on expression of interleukin-2 receptor γ chain (IL-2Rγc), IL-15, and the major histocompatibility complex (MHC) class Ib protein H2-T3, also known as the thymus leukemia antigen or TL. While lineage tracking experiments indicated that iCD8α cells have a lymphoid origin, their development was independent of the transcriptional suppressor Id2, suggesting that these cells do not belong to the family of innate lymphoid cells. Finally, we identified cells with a similar phenotype in humans, which were profoundly depleted in newborns with necrotizing enterocolitis. These findings suggest a critical role of iCD8α cells in immune responses associated with the intestinal epithelium.

Mucosal memory CD8+ T cells are selected in the periphery by an MHC class I molecule

The presence of immune memory at pathogen entry sites is a prerequisite for protection. Nevertheless, the mechanisms that warrant immunity at peripheral interfaces are not understood. Here we show that the non-classical MHC class I molecule, the thymus leukemia antigen (TL), induced on dendritic cells interacting with CD8αα on activated CD8αβ+T cells, mediated affinity-based selection of memory precursor cells. Furthermore, constitutive expression of TL on epithelial cells led to continued selection of mature CD8αβ memory T cells. The TL-CD8αα-driven memory process was essential for the generation of memory CD8αβ T cells in the intestine and accumulation of highly antigen sensitive CD8αβ memory T cells that form the first line of defense at the largest entry port for pathogens.

Intestinal epithelial cells modulate CD4 T cell responses via the thymus leukemia antigen (161.8)

Abstract The thymus leukemia (TL) antigen is a non-classical MHC class I molecule primarily expressed on intestinal epithelial cells (IEC) that interacts with CD8αα, which is primarily expressed by intestinal intraepithelial lymphocytes. The close proximity of cells expressing these two molecules suggests that TL-expressing IEC can regulate CD8αα-expressing lymphocytes. Here, we report that TL influences the cytokine profile and pathogenic potential of CD4+ T cells expressing CD8αα. We show that TL+ IEC regulates IL-17 production by CD4+ T cells. Consequently, TL expression enhances development of colitis in an adoptive transfer model and confers resistance to Citrobacter rodentium infection by allowing the generation of an adequate Th17 response. Thus, TL expressed on IEC modulates CD4+ T cell responses in the intestinal mucosa.

Tissue-Specific Cues Direct Fate Decisions of Mucosal Memory T Cells (39.21)

Abstract Mucosal memory CD8 T cells are characterized by their location at peripheral interfaces and the ability to immediately exhibit effector functions. By contrast, lymphoid memory T cells reside within lymphoid tissues and require further differentiation to become effectors. Both memory subtypes contribute to protective immunity, but the accumulation of mucosal effector memory T cells at pathogen portals forms a central component of protective immunity. The process that drives the compartmentalization and functional differentiation of this memory subset is poorly understood. Here we show that the thymus leukemia (TL) antigen, a ligand for the TCR co-repressor, CD8αα, serves a key role in the molecular mechanism that determines the fate of mucosal CD8αβ+ effector T cells. TL specifically controls the survival of CD8αα+CD8αβ+ effectors, but eliminates CD8αα negative CD8αβ effectors. This TL-CD8αα-mediated process allows the intestinal environment to selectively preserve the "fittest" effector cells to become mucosal memory T cells that form the first line of immune protection.

The thymus leukemia antigen controls intraepithelial lymphocyte function and inflammatory bowel disease (39.53)

Abstract Intestinal intraepithelial lymphocytes (IEL) bear a partially activated phenotype that permits them to rapidly respond to antigenic insults. However, this phenotype also implies that IEL must be highly controlled to prevent misdirected immune reactions. It has been suggested that IEL are regulated through the interaction of the CD8alpha-alpha homodimer with the thymus leukemia (TL) antigen expressed by intestinal epithelial cells. We have generated and characterized mice genetically deficient in TL expression. Our findings show that TL expression plays a critical role in maintaining IEL effector functions. Moreover, TL deficiency accelerated colitis in a genetic model of inflammatory bowel disease. These findings reveal an important regulatory role of TL in controlling IEL function and intestinal inflammation.

Thymus leukemia antigen controls intraepithelial lymphocyte function and inflammatory bowel disease

Intestinal intraepithelial lymphocytes (IEL) bear a partially activated phenotype that permits them to rapidly respond to antigenic insults. However, this phenotype also implies that IEL must be highly controlled to prevent misdirected immune reactions. It has been suggested that IEL are regulated through the interaction of the CD8alpha alpha homodimer with the thymus leukemia (TL) antigen expressed by intestinal epithelial cells. We have generated and characterized mice genetically-deficient in TL expression. Our findings show that TL expression has a critical role in maintaining IEL effector functions. Also, TL deficiency accelerated colitis in a genetic model of inflammatory bowel disease. These findings reveal an important regulatory role of TL in controlling IEL function and intestinal inflammation.

Immunity against mouse thymus‐leukemia antigen (TL) protects against development of lymphomas induced by a chemical carcinogen, N‐butyl‐N‐nitrosourea

Mouse thymus-leukemia antigens (TL) are aberrantly expressed on T lymphomas in C57BL/6 (B6) and C3H/He (C3H) mice, while they are not expressed on normal T lymphocytes in these strains. When N-butyl-N-nitrosourea (NBU), a chemical carcinogen, was administered orally to B6 and C3H strains, lymphoma development was slower than in T3(b)-TL gene-transduced counterpart strains expressing TL ubiquitously as self-antigens, suggesting that anti-TL immunity may play a protective role. In addition, the development of lymphomas was slightly slower in C3H than in B6, which seems to be in accordance with the results of skin graft experiments indicating that both cellular and humoral immunities against TL were stronger in C3H than B6 mice. The interesting finding that B lymphomas derived from a T3(b)-TL transgenic strain (C3H background) expressing a very high level of TL were rejected in C3H, but not in H-2K(b) transgenic mice (C3H background), raises the possibility that TL-specific effector T cell populations are eliminated and/or energized to a certain extent by interacting with H-2K(b) molecules.

The TL MHC class Ib molecule has only marginal effects on the activation, survival and trafficking of mouse small intestinal intraepithelial lymphocytes.

Thymus leukemia antigen (TL) is an MHC class Ib molecule that is highly conserved in rats and mice with no obvious human homolog. TL is expressed in mouse small intestinal epithelial cells and is known to interact with CD8alphaalpha homodimers, which are expressed by intraepithelial lymphocytes (IELs), some other T cell subsets and some non-T cells such as a subset of dendritic cells. We show here that TL is abundantly expressed on the basolateral surface of mouse small intestinal epithelial cells and that expression is abrogated in beta2m-/- mice but unaffected in TCR-/- mice or CD8alpha chain-/- mice. We demonstrate that the interaction between TL and CD8alphaalpha is not necessary for IEL survival in vitro or in vivo and does not modulate IEL trafficking in vivo. TL co-stimulation of alpha-CD3 antibody-activated IELs resulted in modestly enhanced production of IFN-gamma in one subset of IELs. The lack of effect on IEL survival and trafficking and the modest effect on IFN-gamma production suggest that the functional consequences of TL interaction with CD8alphaalpha as well as the more general biological role of TL in mucosal immunity remains to be discovered.

Thymus-leukemia antigen (TL) as a major histocompatibility complex (MHC) class Ib molecule and tumor-specific antigen.

Mouse thymus-leukemia antigens (TL) belong to the family of major histocompatibility complex (MHC) class Ib antigens and have a unique mode of expression, i.e., in contrast to other MHC class Ib or Ia antigens, they are found restricted to the intestines in all mouse strains, but also in the thymus of certain strains (TL(+) strains). Nevertheless, a proportion of T lymphomas/leukemias in strains that do not express TL in the thymus (TL(-) strains) feature TL as a tumor antigen. TL was originally defined serologically, but subsequently we have succeeded in generating T cell receptor (TCR) and cytotoxic T lymphocytes (CTL) recognizing TL. By use of TL tetramers free from peptides and transfectants expressing various TL/H-2 chimeric molecules, we have been able to show that TL-specific CTL recognize the alpha1/alpha2 domain of TL without any additional antigen molecules. We previously reported that one of TL's functions in the thymus is positive selection of TCR CTL. Recent studies with TL tetramers revealed that they can bind to normal intestinal intraepithelial lymphocytes (iIEL) and thymocytes in a CD8-dependent, but TCR/CD3-independent manner, while their binding to TL-specific CTL is TCR/CD3- and CD8-dependent. The possible significance of these findings in relation to the roles of TL in the intestines is discussed. We have long been interested in TL as a model tumor antigen which shares characteristics with human differentiation tumor antigens, and we have demonstrated that growth of TL(+) lymphoma cells in vivo is suppressed by immunization with TL(+) skin or dendritic cells (DC) from TL transgenic mice. In addition, anti-tumor effects against TL(+) T lymphomas were obtained by adoptive transfer of TL tetramer strongly-positive TL-specific CTLs.

Thymus leukemia antigen (TL)-specific cytotoxic T lymphocytes recognize the alpha1/alpha2 domain of TL free from antigenic peptides.

The thymus leukemia antigens (TL) belong to the MHC class Ib family and can be recognized by CD8-dependent or -independent cytotoxic T lymphocytes (CTL) showing TL, but not H-2, restriction. We previously reported that the CTL epitope is TAP independent and in the present study we further characterize the recognition mechanism of CD8-dependent TL-specific TCRalphabeta CTL. We first prepared empty TL tetramers by way of peptide-independent folding with recombinant proteins produced in an Escherichia coli expression system, and showed that TL-specific CTL recognized TL without putative TL-associated peptide and/or post-translational modifications of TL by mammalian and insect cells. We next prepared transfectants expressing various chimeric TL molecules with mouse or human MHC class I as well as chimeric TL tetramers with recombinant proteins produced by insect cells, and demonstrated that chimeric TL whose alpha3 domain was replaced by that of H-2K(b), but not of HLA-A2, was sufficient for binding and activation of TL-specific CTL. These results indicate that TL-specific CTL recognize predominantly their alpha1/alpha2 domain as an epitope(s) and that the binding activity to the murine CD8 of the alpha3 domain of H-2K(b) is sufficient to induce their CTL activity, although it is known to be weaker than that of TL, but stronger than that of HLA. The results taken together indicate that CD8-dependent TL-specific TCRalphabeta CTL recognize an epitope(s) of the alpha1/alpha2 domain of TL free from antigenic molecules, and that CD8 plays an important role in stable interactions between TL and their corresponding TCR.

T cell responses modulated through interaction between CD8alphaalpha and the nonclassical MHC class I molecule, TL.

The thymus leukemia antigen (TL) is a nonclassical class I molecule, expressed abundantly on intestinal epithelial cells. We show that, in contrast to other major histocompatibility complex (MHC) class I molecules that bind CD8alphabeta, TL preferentially binds the homotypic form of CD8alpha (CD8alphaalpha). Thus, TL tetramers react specifically to CD8alphaalpha-expressing cells, including most intestinal intraepithelial lymphocytes. Compared with CD8alphabeta, which recognizes the same MHC as the T cell receptor (TCR) and thus acts as a TCR coreceptor, high-affinity binding of CD8alphaalpha to TL modifies responses mediated by TCR recognition of antigen presented by distinct MHC molecules. These findings define a novel mechanism of lymphocyte regulation through CD8alphaalpha and MHC class I.

The binding of thymus leukemia (TL) antigen tetramers to normal intestinal intraepithelial lymphocytes and thymocytes.

Thymus leukemia (TL) Ags belong to the family of nonclassical MHC class I Ags and can be recognized by both TCRalphabeta and TCRgammadelta CTL with TL, but not H-2 restriction. We previously reported that the CTL epitope is TAP independent, but the antigenic molecule(s) presented by TL has yet to be determined. In the present study, TL tetramers were prepared with T3(b)-TL and murine beta(2)-microglobulin, not including antigenic peptides, and binding specificity was studied. CTL clones against TL Ags were stained with the T3(b)-TL tetramer, and the binding shown to be CD3 and CD8 dependent. Normal lymphocytes from various origins were also studied. Surprisingly, most CD8(+) intraepithelial lymphocytes derived from the small intestines (iIEL), as well as CD8(+) and CD4(+)CD8(+) thymocytes, were stained, while only very minor populations of CD8(+) cells derived from other peripheral lymphoid tissues, such as spleen and lymph nodes, were positive. The binding of T3(b)-TL tetramers to CD8(+) iIEL and thymocytes was CD8 dependent, but CD3 independent, in contrast to that to TL-restricted CTL. These results altogether showed that TL-restricted CTL can be monitored by CD3-dependent binding of T3(b)-TL tetramers. In addition, CD3-independent T3(b)-TL tetramer binding to iIEL and thymocytes may imply that TL expressed on intestinal epithelium and cortical thymocytes has a physiological function interacting with these tetramer(+)CD8(+) T lymphocytes.

The epitope detected by cytotoxic T lymphocytes against thymus leukemia (TL) antigen is TAP independent.

Thymus leukemia (TL) antigens belong to the family of MHC class Ib antigens. We have shown in our previous studies that they serve as transplantation antigens, and can be recognized by both TCR alpha beta and TCR gamma delta cytotoxic T lymphocytes (CTL) with TL but not H-2 restriction. Although TL are known to be expressed TAP independently, it is unclear whether peptide loading on TL molecules is necessary for the formation of CTL epitopes. In the present study, we first showed that TL expression is beta(2)-microglobulin (beta(2)m)-dependent but TAP1 independent by flow cytometric analysis of thymocytes from beta(2)m- or TAP1-deficient mice crossed with TL transgenic mice expressing Tla(a)-3-TL on their thymocytes. Subsequently, we investigated the epitope recognized by CTL derived from C3H mice immunized with skin from a transgenic mouse expressing T3(b)-TL ubiquitously. Bulk CTL lines against TL from primary mixed lymphocyte cultures showed comparable cytotoxicity against T3(b)-TL transfectants of TAP2-deficient murine RMA-S grown at 37 degrees C to that against those grown at 25 degrees C. Furthermore, TCR alpha beta and TCR gamma delta CTL clones against TL recognized TL expressed on T3(b)-TL transfectants of RMA-S and Drosophila melanogaster cells having broad defects in peptide loading of MHC, and lysed these target cells. These results together indicate that TL-specific CTL populations primarily recognize epitopes expressed TAP independently.

Requirement of CD4 T cells for skin graft rejection against thymus leukemia (TL) antigen and multiple epitopes on the TL molecule recognized by CD4 T cells.

By selective depletion of CD4 and CD8 T cells in vivo using the respective mAbs, we demonstrate that CD4 T cells are necessary for skin graft rejection against thymus leukemia (TL) Ag. The skin expressing T3b-TL Ag from transgenic C3H Tg.Con.3-1 mice given chimeric H-2Kb/T3b-TL gene was rejected when grafted onto C3H/He recipient mice. Depletion of CD4, but not of CD8, T cells blocked rejection. CD8 CTL were generated in MEM (control)-treated C3H/He recipient mice, while Thy-1+ CD4- CD8- CTL were generated in CD8-depleted recipient mice after rejection. However, no CTL were generated in CD4-depleted or both CD4- and CD8-depleted recipient mice. Thus, the generation of both CD8 and Thy-1+ CD4- CD8- CTL was dependent on CD4 T cells. Ab blocking indicated that both CD8 and Thy-1+ CD4- CD8- CTL were TCR alphabeta and recognized TL Ag. We furthermore demonstrated that CD4 T cells in spleen cells from C3H/He mice that had rejected C3H Tg.Con.3-1 skin showed a weak, but significant, proliferative response to in vitro stimulation with mitomycin C-treated C3H Tg.Con.3-1 spleen cells. Analysis of the reactivity of bulk CD4 T cell lines to 73 synthetic overlapping peptides encompassing the entire T3b-TL molecule showed that CD4 T cells recognized multiple epitopes on the T3b-TL molecule in an APC-dependent manner.

Transgenic Mice Expressing the Thymus Leukemia Antigen Fail to Control Cutaneous Herpes Simplex Virus Infection

TL-transgenic mice expressing the thymus leukemia antigen demonstrate a lack of viral clearance following cutaneous HSV infection of the footpad. In this study, both uninfected and HSV-infected TL-trans- genic mice demonstrate increased concentrations of IL-4 as well as decreased concentrations of IFN-γ which may possibly underlie the impairment of viral clearance. Furthermore, lymphocytes from HSV-infected nontransgenic mice, adoptively transferred into HSV-infected TL-transgenic mice, promoted viral clearance and led to an increase in IFN-γ production. Transgenic mice which were subcutaneously injected with IFN-γ in the right footpad were also capable of clearing the viral challenge; however, clearance was restricted solely to the right footpad. These studies support the possibility of perturbations in the immune system of TL-transgenic mice and effectively demonstrate the utility of this model system in the study of HSV clearance, persistence, and potential spontaneous reactivation. Moreover, the TL-transgenic animals may provide a useful model system for additional studies requiring a host system skewed toward a Th2 phenotype.

Expansion of natural (NK1+) T cells that express alpha beta T cell receptors in transporters associated with antigen presentation-1 null and thymus leukemia antigen positive mice.

Thymic selection of natural killer-1+ natural T cells that express alpha beta T cell receptors requires a conserved beta 2-microglobulin-associated molecule, presumably CD1d, displayed by CD4+8+ thymocytes. Here we demonstrate that positive selection of natural T cells occurs independent of transporters associated with antigen presentation-1 (TAP-1) function. Moreover, natural T cells in TAP-1o/o mice are numerically expanded. Several H-2 class Ib molecules function in a TAP-independent manner, suggesting that if expressed in TAP-1o/o thymocytes, they could play a role in natural T cell development. Of these class Ib molecules, H-2TL is expressed by TAP-1o/o thymocytes. Moreover, we find that thymi of TL+ mice congenic or transgenic for H-2T18 also have a numerically expanded natural T cell repertoire compared with TL- mice. This expansion, as in TAP-1o/o thymi, is evident in each of the limited T cell receptor V beta chains expressed by natural T cells, suggesting that TL and CD1d impact similar repertoires. Thus TL, in addition to CD1d, plays a role in natural T cell development.

Thymus-leukemia antigen interacts with T cells and self-peptides.

The functional role of the class Ib thymus-leukemia (TL) Ag expressed within the thymic cortex and intestinal mucosa of the mouse remains unknown. In an approach to elucidate the potential functionality of TL, we developed transgenic mice that ectopically express the H-2T18d gene product on essentially all nucleated cells through the control of a heterologous H-2Kb gene promoter. Transgenic mice demonstrated an increase in the number of CD4+ lymphocytes within the thymus and lymph nodes; these cells displayed an altered T cell receptor repertoire possibly suggesting a role for the ectopically expressed TL protein. The TL protein additionally displayed the characteristics of a bona fide transplantation Ag, because skin grafts from transgenic animals onto MHC- and minor histocompatibility Ag-matched nontransgenic recipient mice resulted in a rapid and vigorous immunologic rejection of the allograft. In MLR studies, transgenic stimulator cells induced the proliferation of responders to a level intermediate between genetically identical and H-2-disparate responder-stimulator combinations. The TL protein was also capable of stimulating cytotoxic T lymphocytes, thereby resulting in specific lysis of TL+ target cells. Further data demonstrated that the TL protein assembles with peptides that are modified at the amino terminus, and that TL retains these molecules at the cell surface. Together, these data suggest that H-2T18d is capable of interacting with T cells via a bound peptide. These data further support the possibility that TL may subserve a specialized function within the immunologic system.

A Novel T-Cell Differentiation Antigen Expressed in Immature Human Thymocytes and Neuroglial Cells

We report a novel human thymocyte differentiation antigen ICT-1 with a molecular weight of 49 kDa that is noncovalently associated with another 12-kDa protein. The ICT-1 antigen is expressed in 50-70% of total thymocytes, but not in resting or PHA-activated peripheral blood T-cells and bone marrow cells. The thymocytes expressing ICT-1 antigen appear after the 18th week of gestation during fetal development. Since the distribution pattern of the ICT-1 antigen within thymus partly overlaps with that of the CD1 antigens, we investigated whether ICT-1 was one of the CD1 antigen family. However, the failure of anti-ICT-1 antibody to react with mouse L cells transfected with cDNA of CD1a, -b, and -c and the different histologic distribution patterns from that of CD1d strongly suggest that the anti-ICT-1 antibody recognizes an antigen distinct from CD1. Furthermore, ICT-1 is also expressed in human neuroglial cells such as oligodendroglioma, glioblastoma multiforme, Ewing's sarcoma, and cerebellar astrocyte. Hence we believe that the ICT-1 antigen may be a novel thymus-leukemia (TL) antigen or a nonclassical MHC class I antigen.