Ada M. Kruisbeek

Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8+ T cells.

Many tumor-associated antigens are derived from nonmutated “self” proteins. T cells infiltrating tumor deposits recognize self-antigens presented by tumor cells and can be expanded in vivo with vaccination. These T cells exist in a functionally tolerant state, as they rarely result in tumor eradication. We found that tumor growth and lethality were unchanged in mice even after adoptive transfer of large numbers of T cells specific for an MHC class I–restricted epitope of the self/tumor antigen gp100. We sought to develop new strategies that would reverse the functionally tolerant state of self/tumor antigen-reactive T cells and enable the destruction of large (with products of perpendicular diameters of >50 mm2), subcutaneous, unmanipulated, poorly immunogenic B16 tumors that were established for up to 14 d before the start of treatment. We have defined three elements that are all strictly necessary to induce tumor regression in this model: (a) adoptive transfer of tumor-specific T cells; (b) T cell stimulation through antigen-specific vaccination with an altered peptide ligand, rather than the native self-peptide; and (c) coadministration of a T cell growth and activation factor. Cells, vaccination, or cyto-kine given alone or any two in combination were insufficient to induce tumor destruction. Autoimmune vitiligo was observed in mice cured of their disease. These findings illustrate that adoptive transfer of T cells and IL-2 can augment the function of a cancer vaccine. Furthermore, these data represent the first demonstration of complete cures of large, established, poorly immunogenic, unmanipulated solid tumors using T cells specific for a true self/tumor antigen and form the basis for a new approach to the treatment of patients with cancer.

E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements

E12 and E47 are two helix-loop-helix transcription factors that arise by alternative splicing of the E2A gene. Both have been implicated in the regulation of immunoglobulin gene expression. We have now generated E2A (-/-) mice by gene targeting. E2A-null mutant mice fail to generate mature B cells. The arrest of B cell development occurs at an early stage, since no immunoglobulin DJ rearrangements can be detected in homozygous mutant mice. While immunoglobulin germline I mu RAG-1, mb-1, CD19, and lambda 5 transcripts are dramatically reduced in fetal livers of E2A (-/-) mice, B29 and mu degrees transcripts are present, but at lower levels. In addition, we show that Pax-5 transcripts are significantly reduced in fetal livers of E2A (-/-) mice. These data suggest a crucial role for E2A products as central regulators in early B cell differentiation.

The CD3γ chain is essential for development of both the TCRαβ and TCRγδ lineages

CD3γ and CD3δ are the most closely related CD3 components, both of which participate in the TCRαβ–CD3 complex expressed on mature T cells. Interestingly, however, CD3δ does not appear to participate functionally in the pre‐T‐cell receptor (TCR) complex that is expressed on immature T cells: disruption of CD3δ gene expression has no effect on the developmental steps controlled by the pre‐TCR. Here we report that in contrast with CD3δ, CD3γ is an essential component of the pre‐TCR. We generated mice selectively lacking expression of CD3γ, in which expression of CD3δ, CD3ϵ, CD3ζ, pTα and TCRβ remained undisturbed. Thus, all components for composing a pre‐TCR are available, with the exception of CD3γ. Nevertheless, T‐cell development is severely inhibited in CD3γ‐deficient mice. The number of cells in the thymus is reduced to <1% of that in normal mice, and the large majority of thymocytes lack CD4 and CD8 and are arrested at the CD44−CD25+ double negative (DN) stage of development. Peripheral lymphoid organs are also practically devoid of T cells, with absolute numbers of peripheral T cells reduced to only 2–5% of those in normal mice. Both TCRαβ and TCRγδ lineages fail to develop effectively in CD3γ‐deficient mice, although absence of CD3γ has no effect on gene rearrangements of the TCRβ, δ and γ loci. Furthermore, absence of CD3γ results in a severe reduction in the level of TCR and CD3ϵ expression at the cell surface of thymocytes and peripheral T cells. The defect in the DN to double positive transition in mice lacking CD3γ can be overcome by anti‐CD3ϵ‐mediated cross‐linking. CD3γ is thus essential for pre‐TCR function.

Branching out to gain control: how the pre-TCR is linked to multiple functions

How is signaling specificity achieved by the pre-TCR during selection of T-cell fate? Like the TCR, this receptor controls many functions, and recent studies define which pathways couple the pre-TCR to the molecular events controlling survival, proliferation, allelic exclusion at the TCRbeta locus, and further differentiation.

Pre-TCR Signaling and Inactivation of p53 Induces Crucial Cell Survival Pathways in Pre-T Cells

Signaling through the pre-TCR is essential for early T cell development and is severely impaired in mice lacking the CD3 gamma chain of the pre-TCR. We here address the molecular mechanisms underlying this defect. Impaired pre-TCR signaling is shown to be associated with a profound increase in the number of apoptotic CD4- CD8- (DN) thymocytes. Introduction of p53 deficiency into CD3 gamma-deficient mice completely reverses the cell survival defect in CD3 gamma-deficient DN thymocytes and rescues the block in pre-T cell differentiation. In addition, the CD4+ CD8+ (DP) compartment is expanded to its normal size. These findings suggest that the pre-TCR regulates progression through the DNA-damage checkpoint of the DN to DP transition by inactivating p53.

C-Type Lectin-Like Molecule-1 A Novel Myeloid Cell Surface Marker Associated with Acute Myeloid Leukemia

Acute myeloid leukemia (AML) has a poor prognosis due to treatment-resistant relapses. A humanized anti-CD33 antibody (Mylotarg) showed a limited response rate in relapsed AML. To discover novel AML antibody targets, we selected a panel of single chain Fv fragments using phage display technology combined with flow cytometry on AML tumor samples. One selected single chain Fv fragment broadly reacted with AML samples and with myeloid cell lineages within peripheral blood. Expression cloning identified the antigen recognized as C-type lectin-like molecule-1 (CLL-1), a previously undescribed transmembrane glycoprotein. CLL-1 expression was analyzed with a human anti-CLL-1 antibody that was generated from the single chain Fv fragment. CLL-1 is restricted to the hematopoietic lineage, in particular to myeloid cells present in peripheral blood and bone marrow. CLL-1 is absent on uncommitted CD34+/CD38− or CD34+/CD33− stem cells and present on subsets of CD34+/CD38+ or CD34+/CD33+ progenitor cells. CLL-1 is not expressed in any other tissue. In contrast, analysis of primary AMLs demonstrated CLL-1 expression in 92% (68 of 74) of the samples. As an AML marker, CLL-1 was able to complement CD33, because 67% (8 of 12) of the CD33− AMLs expressed CLL-1. CLL-1 showed variable expression (10–60%) in CD34+ cells in chronic myelogenous leukemia and myelodysplastic syndrome but was absent in 12 of 13 cases of acute lymphoblastic leukemia. The AML reactivity combined with the restricted expression on normal cells identifies CLL-1 as a novel potential target for AML treatment.

Induction of TCR Gene Rearrangements in Uncommitted Stem Cells by a Subset of IL-7 Producing, MHC Class II–Expressing Thymic Stromal Cells

The embryonic thymic microenvironment provides the necessary elements for T cell lineage commitment, but the precise role of individual stromal cell components remains to be determined. Here we address the question of which stromal cell types are required for initiation of V-DJ rearrangements of the TCR-beta and TCR-delta locus in CD117+CD45+ uncommitted fetal liver progenitors. We show that fetal thymic stroma alone is necessary and sufficient for induction of TCR-beta and TCR-delta rearrangements. Furthermore, the ability to induce this T cell commitment step is confined to a subset of MHC class II-positive epithelial cells. Thymic stroma derived from mice with a targeted deletion in the IL-7 gene, however, lacks this ability. These findings set the stage for a further definition of the nature of the thymic stromal cell support in the regulation of T cell commitment.

Mechanisms underlying T-cell tolerance.

To understand how the immune system manages to disarm potentially harmful T cells with reactivity against self-antigens, many immunologists have turned to model systems in which transgenic expression of TCRs, foreign antigens, or both, allows visualization of otherwise undetectable mechanisms. Recent data have offered several new insights into the variables that dictate the mechanism for tolerance employed by peripheral T cells. Some of these mechanisms can also dampen ongoing immune responses against foreign antigens and provide a strikingly powerful supplement to the primary mechanism of tolerance, thymic clonal deletion.

Peptide-Induced Negative Selection of Thymocytes Activates Transcription of an NF-ΚB Inhibitor

Negative selection eliminates thymocytes bearing autoreactive T cell receptors (TCR) via an apoptotic mechanism. We have cloned an inhibitor of NF-kappa B, I kappa BNS, which is rapidly expressed upon TCR-triggered but not dexamethasone- or gamma irradiation-stimulated thymocyte death. The predicted protein contains seven ankyrin repeats and is homologous to I kappa B family members. In class I and class II MHC-restricted TCR transgenic mice, transcription of I kappa BNS is stimulated by peptides that trigger negative selection but not by those inducing positive selection (i.e., survival) or nonselecting peptides. I kappa BNS blocks transcription from NF-kappa B reporters, alters NF-kappa B electrophoretic mobility shifts, and interacts with NF-kappa B proteins in thymic nuclear lysates following TCR stimulation. Retroviral transduction of I kappa BNS in fetal thymic organ culture enhances TCR-triggered cell death consistent with its function in selection.

Early Growth Response Transcription Factors Are Required for Development of CD4−CD8− Thymocytes to the CD4+CD8+ Stage

Progression of immature CD4−CD8− thymocytes beyond the β-selection checkpoint to the CD4+CD8+ stage requires activation of the pre-TCR complex; however, few of the DNA-binding proteins that serve as molecular effectors of those pre-TCR signals have been identified. We demonstrate in this study that members of the early growth response (Egr) family of transcription factors are critical effectors of the signals that promote this developmental transition. Specifically, the induction of three Egr family members (Egr1, 2, and 3) correlates with pre-TCR activation and development of CD4−CD8− thymocytes beyond the β-selection checkpoint. Enforced expression of each of these Egr factors is able to bypass the block in thymocyte development associated with defective pre-TCR function. However, Egr family members may play somewhat distinct roles in promoting thymocyte development, because there are differences in the genes modulated by enforced expression of particular Egr factors. Finally, interfering with Egr function using dominant-negative proteins disrupts thymocyte development from the CD4−CD8− to the CD4+CD8+ stage. Taken together, these data demonstrate that the Egr proteins play an essential role in executing the differentiation program initiated by pre-TCR signaling.