Daniel L. Mueller

Blocked signal transduction to the ERK and JNK protein kinases in anergic CD4 + T cells

T cells activated by antigen receptor stimulation in the absence of accessory cell-derived costimulatory signals lose the capacity to synthesize the growth factor interleukin-2 (IL-2), a state called clonal anergy. An analysis of CD3- and CD28-induced signal transduction revealed reduced ERK and JNK enzyme activities in murine anergic T cells. The amounts of ERK and JNK proteins were unchanged, and the kinases could be fully activated in the presence of phorbol 12-myristate 13-acetate. Dephosphorylation of the calcineurin substrate NFATp (preexisting nuclear factor of activated T cells) also remained inducible. These results suggest that a specific block in the activation of ERK and JNK contributes to defective IL-2 production in clonal anergy.

Mechanisms maintaining peripheral tolerance

The presentation of self-peptide–MHC complexes in the periphery to potentially autoreactive T cells that have escaped negative selection in the thymus poses an important problem to the immune system. In this review, I discuss data that reveal barriers preventing peripheral T cell recognition of self-peptide–MHC complexes, as well as the physiological mechanisms that ensure the elimination or functional inactivation (anergy) of T cells that do come to recognize self-peptide–MHC and threaten the health of the individual.

E3 ubiquitin ligases as T cell anergy factors

E3 ubiquitin ligases have emerged as key molecular regulators of immune cell function. Three families of proteins with ubiquitin ligase activity have been described (the HECT, RING and U-box proteins), and each may be involved in the regulation of immune responses during infection by targeting specific inhibitory molecules for proteolytic destruction. Several HECT and RING E3 proteins have now also been linked to the induction and maintenance of immune self-tolerance: c-Cbl, Cbl-b, GRAIL, Itch and Nedd4 each negatively regulate T cell growth factor production and proliferation. This review will discuss the relationship between the ubiquitination of select components of the antigen-sensing signaling apparatus in T cells and the development and maintenance of the clonal anergy state.

Gene Regulation and Chromatin Remodeling by IL-12 and Type I IFN in Programming for CD8 T Cell Effector Function and Memory

A third signal that can be provided by IL-12 or type I IFN is required for differentiation of naive CD8 T cells responding to Ag and costimulation. The cytokines program development of function and memory within 3 days of initial stimulation, and we show here that programming involves regulation of a common set of ∼355 genes including T-bet and eomesodermin. Much of the gene regulation program is initiated in response to Ag and costimulation within 24 h but is then extinguished unless a cytokine signal is available. Histone deacetylase inhibitors mimic the effects of IL-12 or type I IFN signaling, indicating that the cytokines relieve repression and allow continued gene expression by promoting increased histone acetylation. In support of this, increased association of acetylated histones with the promoter loci of granzyme B and eomesodermin is shown to occur in response to IL-12, IFN-α, or histone deacetylase inhibitors. Thus, IL-12 and IFN-α/β enforce in common a complex gene regulation program that involves, at least in part, chromatin remodeling to allow sustained expression of a large number of genes critical for CD8 T cell function and memory.

Frequent aberrant immunoglobulin gene rearrangements in pro-B Cells revealed by a bcl-xL transgene

During B lymphocyte development, pro-B cells that fail to rearrange an immunoglobulin heavy (IgH) chain allele productively are thought to undergo developmental arrest and death, but because these cells are short-lived in vivo they are not well characterized. Transgenic mice expressing the apoptosis regulatory gene bcl-xL in the B lineage developed large expansions of pro-B cells in bone marrow. V(D)J rearrangements in the expanded populations were nearly all nonproductive, and DJH rearrangements were enriched for joints in DH reading frame 2 and for aberrant joints with extensive DH or JH deletions. Thus, the death of pro-B cells with failed immunoglobulin rearrangements occurs by apoptosis, and bcl-xL can deliver a strong survival signal at the pro-B stage. This analysis also demonstrated that immunoglobulin gene rearrangement is less precise than previously appreciated.

Self-Reactive B Lymphocytes Overexpressing Bcl-xL Escape Negative Selection and Are Tolerized by Clonal Anergy and Receptor Editing

Self-reactive B cells Tg for both a bcl-xL death inhibitory gene and an Ig receptor recognizing hen egg lysozyme (HEL-Ig) efficiently escaped developmental arrest and deletion in mice expressing membrane-bound self-antigen (mHEL). In response to the same antigen, Tg HEL-Ig B cells not expressing bcl-xL were deleted, while cells expressing bcl-2 were arrested at the immature B stage. Bcl-xL Tg B cells escaping negative selection were anergic in both in vitro and in vivo assays and showed some evidence for receptor editing. These studies suggest that Bcl-x may have a distinct role in controlling survival at the immature stage of B cell development and demonstrate that tolerance is preserved when self-reactive B cells escape central deletion.

Prolonged TCR/CD28 Engagement Drives IL-2-Independent T Cell Clonal Expansion through Signaling Mediated by the Mammalian Target of Rapamycin

Proliferation of Ag-specific T cells is central to the development of protective immunity. The concomitant stimulation of the TCR and CD28 programs resting T cells to IL-2-driven clonal expansion. We report that a prolonged occupancy of the TCR and CD28 bypasses the need for autocrine IL-2 secretion and sustains IL-2-independent lymphocyte proliferation. In contrast, a short engagement of the TCR and CD28 only drives the expansion of cells capable of IL-2 production. TCR/CD28- and IL-2-driven proliferation revealed a different requirement for PI3K and for the mammalian target of rapamycin (mTOR). Thus, both PI3K and mTOR activities were needed for T cells to proliferate to TCR/CD28-initiated stimuli and for optimal cyclin E expression. In contrast, either PI3K or mTOR were sufficient for IL-2-driven cell proliferation as they independently mediated cyclin E induction. Interestingly, rapamycin delayed cell cycle entry of IL-2-sufficient T cells, but did not prevent their expansion. Together, our findings indicate that the TCR, CD28, and IL-2 independently control T cell proliferation via distinct signaling pathways involving PI3K and mTOR. These data suggest that Ag persistence and the availability of costimulatory signals and of autocrine and paracrine growth factors individually shape T lymphocyte expansion in vivo.

T-cell Clonal Anergy

Recent experiments have elucidated two molecular mechanisms that may account for the failure of anergic T cell clones to initiate IL-2 gene transcription following TCR stimulation. First, a block has been identified in the ERK and JNK mitogen-activated protein kinase pathways; the block results from a failure to activate p21ras. It leads to reduced induction of c-Fos and JunB proteins and to a failure to form and phosphorylate the activator protein (AP)-1 heterodimers required for IL-2 gene transcriptional activation. Second, repressor molecules (Nil-2-a and a molecule related to AP-1) have been characterized that dominantly inhibit IL-2 gene transcription.

Molecular mechanisms underlying functional T-cell unresponsiveness

Evidence from tissue culture experiments and in vivo studies indicates that certain forms of antigen presentation render T cells unresponsive to subsequent antigenic stimulation. Great progress has been made recently in understanding the induction, maintenance, and in vivo relevance of this unresponsive state, called clonal anergy.

Murine thymic selection quantified using a unique method to capture deleted T cells

Thymic positive and negative selection events generate a T-cell repertoire that is MHC restricted and self-tolerant. The number of T cells undergoing positive and negative selection in normal mice has never been firmly established. We generated mice that lack the proapoptotic molecule Bim (bcl2l11) together with a Nur77GFP transgene, which allowed the identification and enumeration of T cells that would normally undergo clonal deletion. Using this method, we report the striking observation that six times more cells undergo negative selection than complete positive selection. Seventy-five percent of the negatively selected cells are deleted at the double positive stage in the thymic cortex, compared with 25% at the single positive stage in the medulla. The fact that more thymocytes are highly reactive to MHC than are weakly reactive is inconsistent with a random model of recognition and suggests that T-cell recognition is MHC biased. Furthermore, Bim−/− mice had an increased number of GFPhi cells in the peripheral lymphoid tissue and a corresponding increase in antigen experienced or anergic cell phenotype. Our data also show that the CD4+ T cells that are clonally deleted experienced only slightly stronger T-cell receptor signaling than those that developed into regulatory T cells.