Chemical dissection of the cell cycle: probes for cell biology and anti-cancer drug development.

Cancer cell proliferation relies on the ability of cancer cells to grow, transition through the cell cycle, and divide. To identify novel chemical probes for dissecting the mechanisms governing cell cycle progression and cell division, and for developing new anti-cancer therapeutics, we developed and performed a novel cancer cell-based high-throughput chemical screen for cell cycle modulators. This approach identified novel G1, S, G2, and M-phase specific inhibitors with drug-like properties and diverse chemotypes likely targeting a broad array of processes. We further characterized the M-phase inhibitors and highlight the most potent M-phase inhibitor MI-181, which targets tubulin, inhibits tubulin polymerization, activates the spindle assembly checkpoint, arrests cells in mitosis, and triggers a fast apoptotic cell death. Importantly, MI-181 has broad anti-cancer activity, especially against BRAF(V600E) melanomas.

Congenital high-grade sarcoma presenting as skin nodules and respiratory distress in a neonate.

We report, to our knowledge, the first case of a congenital, widespread, aggressive high-grade sarcoma, presented as multiple skin nodules and respiratory distress in a neonate that had a t(9;22)(q22;q11-12) cytogenetic abnormality suggestive of a more indolent extraskeletal myxoid chondrosarcoma (EMC). EMC is generally thought of as a slow-growing tumor that presents between the fourth and sixth decades of life. Our patient was a 45,XY, t(13;14) newborn who presented at birth with subcutaneous nodules involving the face, scalp, back and extremities, as well as multiple intrathoracic, intraabdominal and intracranial masses. Diagnosis was made using electron microscopy and immunohistochemical and cytogenetic studies. Despite attempts to control rapid growth of lesions using high-dose steroids and cis-retinoic acid, patient's clinical status continued to deteriorate and life support was withdrawn at the 26 day of life.

Global DNA methylation profiling reveals silencing of a secreted form of Epha7 in mouse and human germinal center B-cell lymphomas.

Most human lymphomas originate from transformed germinal center (GC) B lymphocytes. While activating mutations and translocations of MYC, BCL2 and BCL6 promote specific GC lymphoma subtypes, other genetic and epigenetic modifications that contribute to malignant progression in the GC remain poorly defined. Recently, aberrant expression of the TCL1 proto-oncogene was identified in major GC lymphoma subtypes. TCL1 transgenic mice offer unique models of both aggressive GC and marginal zone B-cell lymphomas, further supporting a role for TCL1 in B-cell transformation. Here, restriction landmark genomic scanning was employed to discover tumor-associated epigenetic alterations in malignant GC and marginal zone B-cells in TCL1 transgenic mice. Multiple genes were identified that underwent DNA hypermethylation and decreased expression in TCL1 transgenic tumors. Further, we identified a secreted isoform of EPHA7, a member of the Eph family of receptor tyrosine kinases that are able to influence tumor invasiveness, metastasis and neovascularization. EPHA7 was hypermethylated and repressed in both mouse and human GC B-cell non-Hodgkin lymphomas, with the potential to influence tumor progression and spread. These data provide the first set of hypermethylated genes with the potential to complement TCL1-mediated GC B-cell transformation and spread.

CmC(A/T)GG DNA methylation in mature B cell lymphoma gene silencing.

DNA methylation has been linked to gene silencing in cancer. Primary effusion lymphoma (PEL) and myeloma are lymphoid malignancies that arise from terminally differentiated B cells. Interestingly, PEL do not express immunoglobulins or most B lineage-specific genes. The B cell-specific B29 (Igbeta/CD79b) gene is silenced in PEL and some myelomas but is expressed in other normal and malignant B cells. B29 expression was reactivated in PEL by demethylating and histone deacetylase inhibiting treatments. Bisulfite sequencing revealed two types of DNA methylation in silenced B29 promoters: at conventional CpG and at CC(A/T)GG B29 promoter sites. The pattern of methylated CpG ((m)CpG) and C(m)C(A/T)GG B29 promoter methylation observed was similar to that recently reported for epigenetic silencing of an integrated retrovirus. Methylation of C(m)C(A/T)GG sites in the B29 promoter significantly repressed in vivo transcriptional activity. Also, methylation of a central conserved C(m)CTGG B29 promoter site blocked the binding of early B cell factor. This methylated motif formed DNA-protein complexes with nuclear extracts from all cell types examined. Therefore, C(m)C(A/T)GG methylation may represent an important type of epigenetic marker on mammalian DNA that impacts transcription by altering DNA-protein complex formation.

The COOH-terminal domain of FLI-1 is necessary for full tumorigenesis and transcriptional modulation by EWS/FLI-1.

More than 85% of Ewing's family tumors carry a specific chromosomal translocation that fuses the NH(2) terminus of the EWS gene to the COOH terminus of the FLI1 transcription factor. It has been shown previously that both the transactivation domain encoded by EWS and the DNA binding domain of FLI1 were necessary for transforming cells to anchorage independence. We now report that a COOH-terminal domain in addition to the FLI1 DNA binding domain is necessary to promote cellular transformation. NIH 3T3 cells expressing a COOH-terminal deletion mutant (EWS/FLI1 DeltaC) have a greatly reduced capability to form colonies in soft agar and tumors in severe combined immunodeficient mice. The rate of tumor formation for NIH 3T3 that express EWS/FLI1 DeltaC is 50 days, whereas EWS/FLI1 forms tumors within 22 days. In addition, cells expressing the EWS/FLI1 DeltaC mutant failed to completely demonstrate the round-cell histology that is seen in both Ewing's tumor cell lines and NIH 3T3 cells expressing full-length EWS/FLI1. Northern and microarray analyses were performed to assess the effect of loss of the FLI1 COOH terminus on transcriptional modulation of EWS/FLI1 target genes. We found that although EWS/FLI1 DeltaC up-regulates smaller numbers of genes (21 genes) compared with EWS/FLI1 (34 genes), 41% of the EWS/FLI1 targets were also up-regulated by EWS/FLI1 DeltaC. On the other hand, EWS/FLI1 DeltaC is unable to down-regulate genes (3 genes) as efficiently as EWS/FLI1 (39 genes) with only one target gene repressed by both fusion constructs. Our study indicates that the EWS/FLI1 transcription factor has strong transcriptional activating as well as repressing properties and suggests that transcriptional activation and repression of target genes may occur through biochemically different mechanisms.

TCL1 oncogene expression in B cell subsets from lymphoid hyperplasia and distinct classes of B cell lymphoma.

Activation of the TCL1 oncogene has been implicated in T cell leukemias/lymphomas and recently was associated with AIDS diffuse large B cell lymphomas (AIDS-DLBCL). Also, in nonmalignant lymphoid tissues, antibody staining has shown that mantle zone B cells expressed abundant Tcl1 protein, whereas germinal center (GC; centrocytes and centroblasts) B cells showed markedly reduced expression. Here, we analyze isolated B cell subsets from hyperplastic tonsil to determine a more precise pattern of Tcl1 expression with development. We also examine multiple B cell lines and B lymphoma patient samples to determine whether different tumor classes retain or alter the developmental pattern of expression. We show that TCL1 expression is not affected by Epstein-Barr virus (EBV) infection and is high in naïve B cells, reduced in GC B cells, and absent in memory B cells and plasma cells. Human herpesvirus-8 infected primary effusion lymphomas (PEL) and multiple myelomas are uniformly TCL1 negative, whereas all other transformed B cell lines tested express moderate to abundant TCL1. This observation supports the hypothesis that PEL, like myeloma, usually arise from post-GC stages of B cell development. Tcl1 protein is also detected in most naïve/GC-derived B lymphoma patient samples (23 of 27 [85%] positive), whereas most post-GC-derived B lymphomas lack expression (10 of 41 [24%] positive). These data indicate that the pattern of Tcl1 expression is distinct between naïve/GC and post-GC-derived B lymphomas (P < 0.001) and that the developmental pattern of expression is largely retained. However, post-GC-derived AIDS-DLBCL express TCL1 at a frequency equivalent to naïve/GC-derived B lymphomas in immune-competent individuals (7 of 9 [78%] positive), suggesting that TCL1 down-regulation is adversely affected by severe immune system dysfunction. These findings demonstrate that TCL1 expression in B cell lymphoma usually reflects the stage of B cell development from which they derive, except in AIDS-related lymphomas.

Divergent Ewing's sarcoma EWS/ETS fusions confer a common tumorigenic phenotype on NIH3T3 cells.

Ewing's sarcomas express chimeric transcription factors resulting from a fusion of the amino terminus of the EWS gene to the carboxyl terminus of one of five ETS proteins. While the majority of tumors express EWS/FLI1 fusions, some Ewing's tumors contain variant chimeras such as EWS/ETV1 that have divergent ETS DNA-binding domains. In spite of their structural differences, both EWS/ETS fusions up regulate EAT-2, a previously described EWS/FLI1 target gene. In contrast to EWS/FLI1, NIH3T3 cells expressing EWS/ETV1 cannot form colonies in soft agar though coexpression of a dominant negative truncated ETV1 construct attenuates EWS/FLI1 mediated anchorage independent growth. When EWS/ETV1 or EWS/FLI1 expressing NIH3T3 cells are injected into SCID mice, tumors form more often and faster than with NIH-3T3 cells with empty vector controls. The tumorigenic potency of each EWS/ETS fusion is linked to its C-terminal structure, with the FLI1 C-terminus confering a greater tumorigenic potential than the corresponding ETV1 domain. The resulting EWS/ETV1 and EWS/FLI1 tumors closely resemble each other at both a macroscopic and a microscopic level. These tumors differ greatly from tumors formed by NIH3T3 cells expressing activated RAS. These data indicate that in spite of their structural differences, EWS/ETV1 and EWS/FLI1 promote oncogenesis via similar biologic pathways.

TCL1 oncogene expression in AIDS-related lymphomas and lymphoid tissues.

AIDS-related non-Hodgkin's lymphoma (AIDS NHL) comprises a diverse and heterogeneous group of high-grade B cell tumors. Certain classes of AIDS NHL are associated with alterations in oncogenes or tumor-suppressor genes or infections by oncogenic herpesviruses. However, the clinically significant class of AIDS NHL designated immunoblastic lymphoma plasmacytoid (AIDS IBLP) lacks any consistent genetic alterations. We identified the TCL1 oncogene from a set of AIDS IBLP-associated cDNA fragments generated by subtractive hybridization with non-AIDS IBLP. Aberrant TCL1 expression has been implicated in T cell leukemia/lymphoma development, and its expression also has been seen in many established B cell tumor lines. However, TCL1 expression has not been reported in AIDS NHL. We find that TCL1 is expressed in the majority of AIDS IBLP tumors examined. TCL1 protein expression is restricted to tumor cells in AIDS IBLP tissue samples analyzed with immunohistochemical staining. Hyperplastic lymph node and tonsil also exhibit strong TCL1 protein expression in mantle zone B cells and in rare interfollicular zone cells, whereas follicle-center B cells (centroblasts and centrocytes) show weaker expression. These results establish TCL1 as the most prevalent of all of the surveyed oncogenes associated with AIDS IBLP. They also indicate that abundant TCL1 expression in quiescent mantle zone B cells is down-regulated in activated germinal center follicular B cells in parallel to the known expression pattern of BCL-2. High-level expression in nonproliferating B cells suggests that TCL1 may function in protecting naïve preactivated B cells from apoptosis.

EWS/ETS fusion genes induce epithelial and neuroectodermal differentiation in NIH 3T3 fibroblasts.

Ewing's sarcoma is the least differentiated member of the peripheral primitive neuroectodermal (pPNET) tumor family. Chromosomal translocations involving the EWS gene and five different Ets family transcription factor genes create fusion genes encoding aberrant transcription factors and are implicated in the vast majority of Ewing's sarcoma cases. Here, NIH 3T3 fibroblasts were infected with control (tk-neo or RAS) and two different EWS/ETS-expressing retroviruses. In vitro studies of established polyclonal lines expressing the two EWS/ETS genes, either EWS/FLI1 or EWS/ETV1, showed induction of cytokeratin 15 gene expression. Both fusion genes also caused characteristic gross morphologic, histologic, and ultrastructural changes in NIH 3T3 cells when transformed cell lines were injected into CB-17-scid mice. Native NIH 3T3 cells with a spindled cell morphology were converted to polygonal cells with high nucleo-cytoplasmic ratios that continued to express abundant cytokeratin. Extracellular collagen deposition was abolished, rough endoplasmic reticulum was markedly diminished, and rudimentary cell-cell attachments appeared. Most strikingly, neurosecretory-type dense core granules like those seen in pPNET were now evident. This murine model, created in mesenchyme-derived NIH 3T3 cells, demonstrated new characteristics of both neuroectodermal and epithelial differentiation and resembled small round cell tumors microscopically.

Systemic mast cell disease associated with primary ovarian mixed malignant germ cell tumor.

A 12-year-old girl with a mixed malignant germ cell tumor of the ovary, treated by surgery and chemotherapy, developed systemic mast cell disease (SMCD) approximately 3 months after chemotherapy. Hematologic malignancies have previously been noted in patients with mediastinal germ cell tumors but this is the first report of a primary ovarian germ cell neoplasm associated with SMCD.

Nonclassical behavior of the mouse CD1 class I-like molecule.

The mouse CD1 (mCD1) molecule is a class I-like molecule that is encoded outside of the MHC. We show here that mCD1 shares several properties with Ag-presenting class I molecules, including a requirement for beta2-microglobulin for stable cell-surface expression in T lymphocyte transfectants and thymocytes. mCD1 is also capable of binding to mouse CD8alphabeta heterodimers participating in the activation of CD8+ T cells in a manner similar to classical class I molecules. However, mCD1 surface expression is not decreased at high temperatures in cells that lack the transporter associated with Ag processing (TAP), including both RMA-S and Drosophila melanogaster cells. The data indicate that mCD1 does not require TAP to be expressed in a stable fashion at the cell surface. We speculate that the ability of mCD1 to reach the cell surface in transporter-deficient cells may reflect its ability to present a distinct set of ligands. The properties of mCD1 described here can account, in part, for the selection of the diverse populations of T cells that are known to be mCD1 reactive.

Antigen-presenting function of the mouse CD1 molecule.

CD1 molecules are distantly related to major histocompatibility complex (MHC)-encoded class I molecules, and they are coexpressed with beta2 microglobulin (beta2m). In the mouse, CD1 is expressed by intestinal epithelial cells and also by some cells in spleen and lymph node. We have shown that surface expression of mouse CD1 (mCD1) is not dependent upon a functional transporter associated with antigen processing (TAP). This, and other data, suggest that mCD1 may acquire peptides in an intracellular compartment other than the endoplasmic reticulum, where classical class I molecules bind peptide. mCD1 molecules also are distinct from classical class I molecules with regard to the types of peptides that they bind. We have demonstrated that mCD1 molecules preferentially bind peptides much longer than the 8-9 amino acids typical of the peptides that bind to classical class I molecules. The sequence motif for mCD1 peptide binding is characterized by the presence of bulky and hydrophobic amino acid side chains. We have generated mCD1-restricted and peptide-specific T-cell lines, thereby demonstrating the immunologic relevance of peptide binding to mCD1. The reactive T cells are TCR alphabeta+ and CD8+, a phenotype typical of many lymphocytes in both lymph node and intestinal mucosae. We speculate that mCD1 molecules may be capable of sampling peptides from the gut lumen and presenting them to mucosal T lymphocytes. In this way, they may function in the maintenance of normal mucosal homeostasis, and perhaps also in the induction of systemic tolerance to antigens delivered by the oral route. In summary, CD1 molecules are a novel category of antigen-presenting molecules that have features in common with class I molecules, features in common with class II, and properties distinct from either subset of antigen-presenting molecules. Further studies of the antigen-presenting function of these molecules are certain to yield new insight into immune regulation and perhaps also into the mechanism of oral tolerance.

Cloning of the gene encoding the mouse homologue of the human calcium signal-modulating ligand.

A cDNA clone, representing the mouse homologue of the recently described gene encoding the human calcium signal-modulating ligand, was isolated from a mouse thymus library. This clone exhibits extensive conservation of the primary nucleotide and deduced amino-acid sequences that, when considered with a similar secondary protein structure, transcript size and distribution of expression, suggests a similarity in function.

Antigen-presenting function of the TL antigen and mouse CD1 molecules.

The hallmark of all the nonclassical antigen-presenting molecules, including nonclassical class I and nonclassical class II (Karlsson et al. 1992) molecules, is their lack of polymorphism. It is presumed, therefore, that these nonclassical molecules must have a distinct antigen-presenting function in which polymorphism is not advantageous. In some cases this may involve presentation of a nonpeptide antigen, as has been demonstrated for human CD1b. It is possible that a molecule adapted to present bacterial lipids would remain relatively nonpolymorphic, because a lipid, which is the end product of a complex biosynthetic pathway, is likely to evolve less rapidly than a short stretch of amino acid sequence containing a T-cell epitope. Alternatively, the lack of polymorphism could reflect the presentation by these molecules of relatively invariant peptides, such as those derived from heat shock proteins. It also is possible that a nonpolymorphic molecule could be selected for the presentation of modified peptides. An example of this is the M3 molecule, which can bind even short peptides as long as they have a formylated N-terminus (Fischer Lindahl et al. 1991). Based upon their structural differences, we believe it is likely that the TL antigen and mCD1 are likely to present different types of ligands. The presence in the TL antigen of the conserved amino acids, which in class I normally from hydrogen bonds with peptides, suggests that the TL antigen also can present nanomeric peptides. A peptide antigen-presenting function also is suggested by the expression of the TL antigen by at least one antigen-presenting cell type, the epithelial cell of the intestine, and by the ability of alloreactive T cells to recognize the TL molecule. While we favor the hypothesis that the TL antigen presents peptides, the data cited above do not constitute formal proof of any kind of antigen-presenting function, and it remains possible that the TL antigen does something else. As noted above, no attempts to elucidate the structure of the ligands bound to the TL antigen have so far succeeded, including the screening of bacteriophage display libraries (Castaño, A.R., Miller, J.E., Holcombe, H.R., unpublished data). In contrast, our recent work has demonstrated that mCD1 presents relatively long peptides with a structured motif distinct from classical class I molecules. This mCD1-binding motif, which is present in a wide range of proteins, does not by itself provide a simple explanation for the lack of mCD1 polymorphism and, as noted above, it remains possible that the natural ligand for mCD1 is a nonpeptide structure. Besides their lack of polymorphism, the TL antigen and mCD1 molecules share two additional features in common which might give insight into their their biological role. First, their surface expression does not depend upon the presence of a functional TAP transporter, and they probably can reach the cell surface as empty molecules. Second, both molecules are expressed by epithelial cells in the intestine. This leads to the speculation that these two nonclassical class I molecules could be involved in sampling or uptake of lumenal peptides for their ultimate presentation to cells of the systematic immune system. For example, longer lumenal peptides could be taken up by mCD1, and perhaps by the TL antigen, and then further processed to nonamers for presentation by classical class I molecules. They also could be transported across the epithelial cell by the TL antigen or mCD1 and subsequently presented by either class I or class II molecules expressed by cells in the lamina propria. This sampling or uptake mediated by either the TL antigen or mCD1 could play a role in the induction of immune responses, or more likely perhaps, in the induction of systemic oral tolerance to peptide antigens.(ABSTRACT TRUNCATED)

Nonclassical behavior of the thymus leukemia antigen: peptide transporter-independent expression of a nonclassical class I molecule.

The thymus leukemia (TL) antigen is a major histocompatibility complex-encoded nonclassical class I molecule. Here we present data demonstrating that expression of the TL antigen, unlike other class I molecules, is completely independent of the function of the transporter associated with antigen processing (TAP). The TL antigen is expressed by transfected TAP-2-deficient RMA-S cells when these cells are grown at 37 degrees C. In transfected RMA cells, the kinetics of arrival of TL antigen on the cell surface are similar to those of a classical class I molecule. The kinetics are not altered in TAP-deficient RMA-S cells, demonstrating that surface TL expression in TAP-deficient cells is not due to the stable expression of a few molecules that leak out by a TAP-independent pathway. Soluble TL molecules produced by Drosophila melanogaster cells are highly resistant to thermal denaturation, unlike peptide-free classical class I molecules synthesized by these insect cells. In addition, these soluble TL molecules are devoid of detectable bound peptides. The results demonstrate that the TL antigen is capable of reaching the surface without bound peptide, although acquisition of peptide or some other ligand through a TAP-independent pathway cannot be formally excluded. We speculate that the ability of the TL antigen to reach the cell surface, under conditions in which other class I molecules do not, may be related to a specialized function of the TL molecule in the mucosal immune system, and possibly in the stimulation of intestinal gamma delta T cells.

Structure and function of H-2 T (Tla) region class I MHC molecules.

The T region of the mouse major histocompatibility complex (MHC) encodes a relatively large number of nonclassical or nonpolymorphic class I genes. In BALB/c mice, at least five of these genes are likely to encode a functional class I gene product. Some of these T region products are ubiquitously expressed, while others are expressed by just a few tissues. In the second category, the thymus leukemia (TL) antigen, which is encoded in the T region by T3 and T18 genes, is expressed primarily by intestinal epithelial cells and thymocytes. Inspection of the sequences of the alpha 1 and alpha 2 domains, which could encode a peptide binding site in these molecules, indicates that in several cases conserved amino acids important for peptide binding by classical class I molecules are present, suggesting that these nonclassical class I molecules can bind nonamer peptides. On the other hand, analysis of the sequence of the T10d gene product suggests that it can not bind nonamer peptides in a fashion similar to classical class I molecules. Although there are so far no examples of the recognition of defined peptides in the context of T region gene products, there are several examples of T cell recognition of these class I molecules. Both alpha beta and gamma delta T cell receptors are involved in this recognition. Transgenic mice that over express the TL antigen show a variety of abnormalities in thymocyte differentiation and function, providing some support for the hypothesis that this nonclassical class I molecule plays a role in T-cell differentiation. Despite this, the most likely function for T region encoded and other nonclassical class I gene products is a specialized antigen presenting function, perhaps in restricted anatomic sites or to specialized T-cell populations.

The alpha 3 domain of the Qa-2 molecule is defective for CD8 binding and cytotoxic T lymphocyte activation.

Qa-2 is a nonclassical class I molecule encoded by the Q7 gene within the mouse major histocompatibility complex (MHC). Results from previous experiments on Qa-2, and on a chimeric Ld molecule (LQ3) in which the alpha 3 domain is encoded by Q7b, suggested that the alpha 3 domain of Qa-2 does not carry out the functions typical of the alpha 3 domains in other classical and nonclassical class I antigens. Class I molecules that contain the Qa-2 alpha 3 domain are poorly recognized by primary cytotoxic T lymphocytes (CTLs), and do not function normally in either positive or negative selection in vivo. By employing a cell-cell adhesion assay we demonstrate directly that the Qa-2 alpha 3 domain in the context of the LQ3 hybrid molecule cannot bind to human CD8, although other mouse class I alpha 3 domains bind efficiently. In addition, CD8-dependent CTL-mediated lysis of target cells, in a system which requires mouse CD8-class I alpha 3 domain interactions, is deficient in cells that express the Qa-2 alpha 3 domain. When combined with our earlier work on LQ3 transgenic mice, these results provide additional molecular support for the hypothesis that interaction with CD8 is required for both positive and negative selection of class I restricted T cells in the thymus. As the Qa-2 alpha 3 domain sequence does not differ from the previously defined minimal CD8 binding sequence of other class I molecules, these results also suggest that additional amino acids in the alpha 3 domain must be critical for CD8 binding and CTL activation.

Failed self-tolerance and autoimmunity in IgG anti-DNA transgenic mice.

Transgenic mice were generated that express both the H and L chain genes derived from a hybridoma secreting an IgG2a mAb specific for ds- and ssDNA. This hybridoma is derived from a lupus mouse and can accelerate nephritis in young NZB x NZW F1 female mice and induce clinical nephritis in BALB/c mice. Some transgenic B cells did not exhibit allelic exclusion; they expressed both transgene-derived IgG and endogenous IgM intracellularly. Most of the B cells in transgenic mice expressed endogenous IgM, some of them expressed low levels of IgG on cell membranes. The transgenic mice, created in a strain not prone to SLE, expressed elevated serum IgG anti-DNA, and some developed clinical nephritis. The affinity of the spontaneously secreted IgG antibodies for dsDNA were similar in nephritic NZB x NZW F1 and transgenic mice. In contrast to the nontransgenic littermates, immunization of transgenic mice with murine DNA further enhanced serum levels of IgG anti-DNA in transgenic mice. Therefore, expression of transgene-encoded IgG anti-DNA mainly in the secreted form does not provide the signals necessary for allelic exclusion or self-tolerance. Expression of this Ig is sufficient to induce a mild form of autoimmune disease.

The thymus leukemia antigen binds human and mouse CD8.

The thymus leukemia antigen (TLA) is a class Ib, or 'nonclassical' class I molecule, one of several encoded within the Tla locus of the mouse major histocompatibility complex (MHC). It structurally resembles the H-2K, D, and L class I transplantation antigens, which present processed peptides to cytotoxic T lymphocytes (CTLs). Although their function(s) are unknown, there has been recent speculation concerning the possibility that class Ib molecules may present antigens to T cells that express gamma delta T cell antigen receptors (TCRs). In this report, using both a cell-cell adhesion assay and adhesion of T lymphocyte clones to purified plate-bound TLA, we provide evidence that TLA can bind to both human and mouse CD8. We also show that a chimeric class I molecule containing the peptide antigen binding site of Ld and the alpha 3 domain, transmembrane, and cytoplasmic segments of TLA, can support a CD8-dependent immune response by CTLs. These results demonstrate for the first time binding of a class Ib molecule to CD8 with a functional outcome, as is observed for the class I transplantation antigens. The capacity to interact with CD8 has been conserved despite the extensive sequence divergence of TLA in the peptide antigen binding site, suggesting this interaction is highly significant. TLA is expressed by epithelial cells in the mouse small intestine. As these epithelial cells are in close contact with intestinal intraepithelial lymphocytes that are nearly all CD8+, and many of which express the gamma delta TCR, the data are consistent with the hypothesis that TLA is involved in antigen presentation, perhaps to gamma delta-positive lymphocytes in this site.