T-cell regulation by CD28 and CTLA-4.

Activation of T lymphocytes is thought to require at least two signals, one delivered by the T-cell receptor complex after antigen recognition, and one provided on engagement of co-stimulatory receptors, such as CD28. Recent studies are providing clues as to the specific signalling roles of co-stimulatory receptors. Furthermore, superimposition of inhibitory signals, such as those delivered by cytotoxic T-lymphocyte antigen 4 (CTLA-4), leads to a complex network of positive and negative co-stimulatory signals, the integration of which modulates immune responses.

Epidemiology, outcomes, and mortality predictors of invasive mold infections among transplant recipients: a 10-year, single-center experience.

BACKGROUND: The epidemiology of invasive mold infections (IMI) in transplant recipients differs based on geography, hosts, preventative strategies, and methods of diagnosis. METHODS: We conducted a retrospective observational study to evaluate the epidemiology of proven and probable IMI, using prior definitions, among all adult hematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT) recipients in the era of "classic" culture-based diagnostics (2000-2009). Epidemiology was evaluated before and after an initiative was begun to increase bronchoscopy in HSCT recipients after 2005. RESULTS: In total, 106 patients with one IMI were identified. Invasive aspergillosis (IA) was the most common IMI (69; 65.1%), followed by mucormycosis (9; 8.5%). The overall rate of IMI (and IA) was 3.5% (2.5%) in allogeneic HSCT recipients. The overall incidence for IMI among lung, kidney, liver, and heart transplant recipients was 49, 2, 11, and 10 per 1000 person-years, respectively. The observed rate of IMI among human leukocyte antigen-matched unrelated and haploidentical HSCT recipients increased from 0.6% annually to 3.0% after bronchoscopy initiation (P < 0.05). The 12-week mortality among allogeneic HSCT, liver, kidney, heart, and lung recipients with IMI was 52.4%, 47.1%, 27.8%, 16.7%, and 9.5%, respectively. Among allogeneic HSCT (odds ratio [OR]: 0.07, P = 0.007) and SOT (OR: 0.22, P = 0.05) recipients with IA, normal platelet count was associated with improved survival. Male gender (OR: 14.4, P = 0.007) and elevated bilirubin (OR: 5.7, P = 0.04) were significant predictors of mortality for allogeneic HSCT and SOT recipients with IA, respectively. CONCLUSIONS: During the era of culture-based diagnostics, observed rates of IMI were low among all transplants except lung transplant recipients, with relatively higher mortality rates. Diagnostic aggressiveness and host variables impact the reported incidence and outcome of IMI and likely account for institutional variability in multicenter studies. Definitions to standardize diagnoses among SOT recipients are needed.

Bcl-2 proteins: regulators of apoptosis or of mitochondrial homeostasis?

Programmed cell death (apoptosis) is used by multicellular organisms during development and to maintain homeostasis within mature tissues. One of the first genes shown to regulate apoptosis was bcl-2. Subsequently, a number of Bcl-2-related proteins have been identified. Despite overwhelming evidence that Bcl-2 proteins are evolutionarily conserved regulators of apoptosis, their precise biochemical function remains controversial. Three biochemical properties of Bcl-2 proteins have been identified: their ability to localize constitutively and/or inducibly to the outer mitochondrial, outer nuclear and endoplasmic reticular membranes, their ability to form heterodimers with proteins bearing an amphipathic helical BH3 domain, and their ability to form ion-conducting channels in synthetic membranes. The discovery that mitochondria can play a key part in the induction of apoptosis has focused attention on the role that Bcl-2 proteins may have in regulating either mitochondrial physiology or mitochondria-dependent caspase activation. Here we attempt to synthesize our current understanding of the part played by mitochondria in apoptosis with a consideration of how Bcl-2 proteins might control cell death through an ability to regulate mitochondrial physiology.

Restricted immunoglobulin junctional diversity in neonatal B cells results from developmental selection rather than homology-based V(D)J joining.

The mechanism by which coding ends are joined during immunoglobulin (Ig) recombination is poorly understood. Recently, short sequence similarities (2-6 bp) observed at the ends of certain variable (V), diversity (D), and joining (J) gene segments of Ig have been correlated with limited junctional diversity observed in coding exons assembled from these elements. However, it is unclear whether these sequence homologies play any direct role in favoring coding joint formation by influencing the V(D)J recombination process. In this report, we demonstrate that coding sequence similarities do not influence the position of coding joints during V(D)J recombination in vivo. Instead, during embryonic development, B cells with certain joining products undergo progressive selection. Developmental selection is completed before exposure to external antigens and appears to be determined by the amino acid sequence encoded by the coding joint. We conclude that the nucleotide sequences of the coding regions do not play a major role in directing V(D)J recombination. Instead, we propose that limited Ig junctional diversity results from prenatal developmental selection of B cells based on the protein sequence of their surface Ig antigen-binding site. Sequence identities at the ends of coding segments may have evolved because they increase the likelihood that a selectable antigen-binding site is created during a random recombination process.

Evolutionarily conserved Ets family members display distinct DNA binding specificities.

Members of the Ets family of proto-oncogenes encode sequence-specific transcription factors that bind to a purine-rich motif centered around a conserved GGA trinucleotide. Ets binding sites have been identified in the transcriptional regulatory regions of multiple T cell genes including the T cell receptor alpha and beta (TCR-alpha and -beta) enhancers and the IL-2 enhancer, as well as in the enhancers of several T cell-trophic viruses including Maloney sarcoma virus, human leukemia virus type 1, and human immunodeficiency virus-2. T cells express multiple members of the Ets gene family including Ets-1, Ets-2, GABP alpha, Elf-1, and Fli-1. The different patterns of expression and protein-protein interactions of these different Ets family members undoubtedly contribute to their ability to specifically regulate distinct sets of T cell genes. However, previous studies have suggested that different Ets family members might also display distinct DNA binding specificities. In this report, we have examined the DNA binding characteristics of two Ets family members, Ets-1 and Elf-1, that are highly expressed in T cells. The results demonstrate that the minimal DNA binding domain of these proteins consists of adjacent basic and putative alpha-helical regions that are conserved in all of the known Ets family members. Both regions are required for DNA binding activity. In vitro binding studies demonstrated that Ets-1 and Elf-1 display distinct DNA binding specificities, and, thereby interact preferentially with different naturally occurring Ets binding sites. A comparison of known Ets binding sites identified three nucleotides at the 3' end of these sequences that control the differential binding of the Ets-1 and Elf-1 proteins. These results are consistent with a model in which different Ets family members regulate the expression of different T cell genes by binding preferentially to purine-rich sequences that share a GGA core motif, but contain distinct flanking sequences.

bcl-x exhibits regulated expression during B cell development and activation and modulates lymphocyte survival in transgenic mice.

We have assessed during B cell development, the regulation and function of bcl-x, a member of the bcl-2 family of apoptosis regulatory genes. Here we show that Bcl-xL, a product of bcl-x, is expressed in pre-B cells but downregulated at the immature and mature stages of B cell development. Bcl-xL but not Bcl-2 is rapidly induced in peripheral B cells upon surface immunoglobulin M (IgM) cross-linking, CD40 signaling, or LPS stimulation. Transgenic mice that overexpressed Bcl-xL within the B cell lineage exhibited marked accumulation of peripheral B cells in lymphoid organs and enhanced survival of developing and mature B cells. B cell survival was further increased by simultaneous expression of bcl-xL and bcl-2 transgenes. These studies demonstrate that Bcl-2 and Bcl-xL are regulated differentially during B cell development and activation of mature B cells. Induction of Bcl-xL after signaling through surface IgM and CD40 appears to provide mature B cells with an additional protective mechanism against apoptotic signals associated with antigen-induced activation and proliferation.

Long-term acceptance of major histocompatibility complex mismatched cardiac allografts induced by CTLA4Ig plus donor-specific transfusion.

Allograft rejection is a T cell-dependent process. Productive T cell activation by antigen requires antigen engagement of the T cell receptor as well as costimulatory signals delivered through other T cell surface molecules such as CD28. Engagement of CD28 by its natural ligand B7 can be blocked using a soluble recombinant fusion protein, CTLA4Ig. Administration of CTLA4Ig blocks antigen-specific immune responses in vitro and in vivo, and we have shown that treatment of rats with a 7-d course of CTLA4Ig at the time of transplantation leads to prolonged survival of cardiac allografts (median 30 d), although most grafts are eventually rejected. Here, we have explored additional strategies employing CTLA4Ig in order to achieve long-term allograft survival. Our data indicate that donor-specific transfusion (DST) plus CTLA4Ig can provide effective antigen-specific immunosuppression. When DST is administered at the time of transplantation followed by a single dose of CTLA4Ig 2 d later, all animals had long-term graft survival (> 60 d). These animals had delayed responses to donor-type skin transplants, compared with normal rejection responses to third-party skin transplants. Furthermore, donor-matched second cardiac allografts were well tolerated with minimal histologic evidence of rejection. These data indicate that peritransplant use of DST followed by subsequent treatment with CTLA4Ig can induce prolonged, often indefinite, cardiac allograft acceptance. These results may be clinically applicable for cadaveric organ and tissue transplantation in humans.

Bcl-XL and Bcl-2 repress a common pathway of cell death.

The effect of Bcl-xL upon the developmental death of T cells was assessed by generating transgenic mice that expressed Bcl-xL within all thymocyte subsets. Bcl-xL protected thymocytes from a variety of apoptotic stimuli, including gamma irradiation, glucocorticoids, and anti-CD3 treatment. Bcl-xL altered thymocyte maturation, resulting in increased numbers of CD3int/hi and CD4-8+ thymocytes. Overall, the phenotype of Bcl-xL transgenics was essentially indistinguishable from a Bcl-2 transgenic model. Overexpression of Bcl-xL or Bcl-2 resulted in the down-regulation of the other molecule, providing further evidence of their reciprocal regulation. In a genetic test of redundancy, the Bcl-xL transgene rescued mature T cells in Bcl-2 null mice. Immunoprecipitation indicated that Bcl-xL, like Bcl-2, heterodimerized with the death-promoting molecule Bax in thymocytes. This in vivo model argues that Bcl-xL, like Bcl-2, functions in a common pathway to repress cell death.

Cytotoxic T lymphocyte antigen 4 (CTLA4) blockade accelerates the acute rejection of cardiac allografts in CD28-deficient mice: CTLA4 can function independently of CD28.

Cytotoxic T lymphocyte antigen 4 (CTLA4) appears to negatively regulate T cell activation. One mechanism by which CTLA4 might antagonize T cell function is through inhibition of CD28 signaling by competing for their shared ligands B7-1 and B7-2. In addition, CTLA4 ligation could initiate a signaling cascade that inhibits T cell activation. To address whether CTLA4 could inhibit immune responses in the absence of CD28, rejection of heart allografts was studied in CD28-deficient mice. H-2(q) hearts were transplanted into allogeneic wild-type or CD28-deficient mice (H-2(b)). Graft rejection was delayed in CD28-deficient compared with wild-type mice. Treatment of wild-type recipients with CTLA4-immunoglobulin (Ig), or with anti-B7-1 plus anti-B7-2 mAbs significantly prolonged allograft survival. In contrast, treatment of CD28-deficient mice with CTLA4-Ig, anti-B7-1 plus anti-B7-2 mAbs, or a blocking anti-CTLA4 mAb induced acceleration of allograft rejection. This increased rate of graft rejection was associated with more severe mononuclear cell infiltration and enhanced levels of IFN-gamma and IL-6 transcripts in donor hearts of untreated wild-type and CTLA4-Ig- or anti-CTLA4 mAb-treated CD28-deficient mice. Thus, the negative regulatory role of CTLA4 extends beyond its potential ability to prevent CD28 activation through ligand competition. Even in the absence of CD28, CTLA4 plays an inhibitory role in the regulation of allograft rejection.

Limited role of CD28-mediated signals in T helper subset differentiation.

The role of CD28-mediated signals in T helper cell maturation is not fully understood. We tested the requirement for costimulation through CD28 in several systems of CD4+, T cell differentiation. In vivo priming of mice with genetic disruption of CD28 (CD28-/-) yielded normal levels of antigen-specific interferon gamma production but markedly diminished levels of interleukin 4 (IL-4) after in vitro restimulation. In response to the pathogenic microbe, Leishman a major, C57BL6 CD28-/- mice were fully capable of controlling infection and exhibited a normal T helper 1 response. BALB/c CD28-/- mice unexpectedly exhibited normal susceptibility to L. major. BALB/c CD28-/- mice developed high levels of IL-4 mRNA and protein induction in the draining lymph nodes. In addition, susceptibility of BALB/c CD28-/- mice was reversed by neutralization of IL-4 in vivo. We also activated transgenic CD28-bearing T cells from the BALB and C57BL background in vitro in the presence of CTLA4Ig. BALB cells had greater IL-4 producing capacity than C57BL cells in the absence of costimulation. Diverse factors including costimulatory signals, genetic polymorphism, and the nature of the immunogen all influence T helper phenotype commitment, but these results provide evidence that CD28 is not an absolute requirement for generating either Th1 or Th2 responses.

CD43 is a murine T cell costimulatory receptor that functions independently of CD28.

Costimulation mediated by the CD28 receptor has been shown to play an important role in the development of a vigorous T cell immune response. Nevertheless, CD28-deficient mice can mount effective T cell-dependent immune responses. These data suggest that other costimulatory molecules may play a role in T cell activation. In a search for other costimulatory receptors on T cells, we have characterized a monoclonal antibody (mAb) that can costimulate T cells in the absence of accessory cells. Similar to CD28 antibodies, this mAb, R2/60, was found to synergize with T cell receptor engagement in inducing proliferation. Independent ligation of CD3 and the ligand recognized by R2/60 results in T cell proliferation, suggesting that the two molecules do not have to colocalize to activate the R2/60 costimulatory pathway. R2/60 does not react with CD28, and furthermore, R2/60 costimulates in a CD28-independent fashion since the mAb costimulates T cells from the CD28-deficient mice as well as wild-type mice. Expression cloning of the R2/60 antigen identified the ligand as murine CD43. Together, these data demonstrate that CD43 can serve as a receptor on T cells that can provide CD28-independent costimulation.

Autoreactive cytotoxic T lymphocytes in human immunodeficiency virus type 1-infected subjects.

A subtractive analysis of peptides eluted from major histocompatibility complex (MHC) class I human histocompatibility leukocyte antigen (HLA)-A2.1 molecules purified from either human immunodeficiency virus type-1 (HIV-1)-infected or uninfected cells was performed using micro high-performance liquid chromatography and mass spectrometry. Three peptides unique to infected cells were identified and found to derive from a single protein, human vinculin, a structural protein not known to be involved in viral pathogenesis. Molecular and cytofluorometric analyses revealed vinculin mRNA and vinculin protein overexpression in B and T lymphocytes from HIV-1-infected individuals. Vinculin peptide-specific CTL activity was readily elicited from peripheral blood lymphocytes of the majority of HLA-A2.1+, HIV+ patients tested. Our observations suggest that atypical vinculin expression and MHC class I-mediated presentation of vinculin-derived peptides accompany HIV infection of lymphoid cells in vivo, with a resultant induction of antivinculin CTL in a significant portion of HIV+ (HLA-A2.1+) individuals.

Antibody and B7/BB1-mediated ligation of the CD28 receptor induces tyrosine phosphorylation in human T cells.

CD28 is an adhesion receptor expressed as a 44-kD dimer on the surface of a major subset of human T cells. The CD28 receptor regulates the production of multiple lymphokines, including interleukin 2 (IL-2), by activation of a signal transduction pathway that is poorly understood. Here we show that ligation of CD28 by a monoclonal antibody (mAb) or by a natural ligand, B7/BB1, induces protein tyrosine phosphorylation that is distinct from T cell receptor (TCR)-induced tyrosine phosphorylation. CD28-induced protein tyrosine phosphorylation was greatly enhanced in cells that had been preactivated by ligation of the TCR, or by pretreatment with phorbol esters. Rapid and prolonged tyrosine phosphorylation of a single substrate, pp100, was induced in T cells after interaction with B7/BB1 presented on transfected Chinese hamster ovary (CHO) cells. Anti-B7 mAb inhibited B7/BB1 receptor-induced tyrosine phosphorylation, indicating that B7-CD28 interaction was required. CD28-induced tyrosine phosphorylation was independent of the TCR because it occurred in a variant of the Jurkat T cell line that does not express the TCR. Herbimycin A, a protein tyrosine kinase inhibitor, could prevent CD28-induced tyrosine phosphorylation and CD28-induced IL-2 production in normal T cells. The simultaneous crosslinking of CD28 and CD45, a tyrosine phosphatase, could prevent tyrosine phosphorylation of pp100. These results suggest that specific tyrosine phosphorylation, particularly of pp100, occurs directly as a result of CD28 ligand binding and is involved in transducing the signal delivered through CD28 by accessory cells that express the B7/BB1 receptor. Thus, this particular form of signal transduction may be relevant to lymphokine production and, potentially may provide a means to study the induction of self-tolerance, given the putative role of the costimulatory signal in the induction of T cell activation or anergy.

Induction of cytotoxic T lymphocyte antigen 4 (CTLA-4) restricts clonal expansion of helper T cells.

Cytotoxic T lymphocyte antigen (CTLA)-4 plays an essential role in immunologic homeostasis. How this negative regulator of T cell activation executes its functions has remained controversial. We now provide evidence that CTLA-4 mediates a cell-intrinsic counterbalance to restrict the clonal expansion of proliferating CD4(+) T cells. The regulation of CTLA-4 expression and function ensures that, after approximately 3 cell divisions of expansion, most progeny will succumb to either proliferative arrest or death over the ensuing three cell divisions. The quantitative precision of the counterbalance hinges on the graded, time-independent induction of CTLA-4 expression during the first three cell divisions. In contrast to the limits imposed on unpolarized cells, T helper type 1 (Th1) and Th2 effector progeny may be rescued from proliferative arrest by interleukin (IL)-12 and IL-4 signaling, respectively, allowing appropriately stimulated progeny to proceed to the stage of tissue homing. These results suggest that the cell-autonomous regulation of CTLA-4 induction may be a central checkpoint of clonal expansion of CD4(+) T cells, allowing temporally and spatially restricted growth of progeny to be dictated by the nature of the threat posed to the host.

Bcl-2-family proteins: the role of the BH3 domain in apoptosis.

Bcl-2-related proteins have come to occupy a prominent position in the realm of programmed cell death. Members of this fast-growing family are highly related in one or more specific regions, commonly referred to as Bcl-2 homology (BH) domains. BH domains contribute at multiple levels to the function of these proteins in cell death and survival. Particularly intriguing is the emergence of the BH3 domain as a potent 'death domain' and of a growing subclass of pro-apoptotic proteins with no similarity to Bcl-2 beyond their BH3 homology. Here, the authors classify proteins of the Bcl-2 family on the basis of function and domain organization, discuss the importance of the BH3 domain in protein-protein interactions and in cell death and provide possible explanations for the perceived redundancy in the expression of this subclass of death promoters.

Apoptosis in the pathogenesis and treatment of disease.

In multicellular organisms, homeostasis is maintained through a balance between cell proliferation and cell death. Although much is known about the control of cell proliferation, less is known about the control of cell death. Physiologic cell death occurs primarily through an evolutionarily conserved form of cell suicide termed apoptosis. The decision of a cell to undergo apoptosis can be influenced by a wide variety of regulatory stimuli. Recent evidence suggests that alterations in cell survival contribute to the pathogenesis of a number of human diseases, including cancer, viral infections, autoimmune diseases, neurodegenerative disorders, and AIDS (acquired immunodeficiency syndrome). Treatments designed to specifically alter the apoptotic threshold may have the potential to change the natural progression of some of these diseases.

Differential expression of c-myb mRNA in murine B lymphomas by a block to transcription elongation.

Expression of c-myb proto-oncogene messenger RNA (mRNA) and protein has been detected principally in tumors and in normal tissue of hematopoietic origin. In each hematopoietic lineage examined, expression of the c-myb gene is markedly downregulated during hematopoietic maturation. However, the mechanism by which differential expression of the c-myb gene is regulated is not known. In murine B-lymphoid tumor cell lines, the amount of steady-state c-myb mRNA is 10 to more than 100 times greater in pre-B cell lymphomas than in B cell lymphomas and plasmacytomas. The downregulation of c-myb mRNA correlates with events at the pre-B cell-B cell junction. Differential expression of c-myb mRNA levels detected between a pre-B cell lymphoma and a mature B cell lymphoma is now shown to be mediated by a block to transcription elongation in the first intron of the c-myb locus. In addition, this developmentally regulated difference in transcriptional activity is correlated with alterations in higher order chromatin structure as reflected by changes in the patterns of hypersensitivity to deoxyribonuclease I at the 5' end of the c-myb transcription unit. Regulation of transcription elongation may provide a more sensitive mechanism for rapidly increasing and decreasing mRNA levels in response to external stimuli than regulation of the initiation of transcription.

Regulation of the Ets-related transcription factor Elf-1 by binding to the retinoblastoma protein.

The retinoblastoma gene product (Rb) is a nuclear phosphoprotein that regulates cell cycle progression. Elf-1 is a lymphoid-specific Ets transcription factor that regulates inducible gene expression during T cell activation. In this report, it is demonstrated that Elf-1 contains a sequence motif that is highly related to the Rb binding sites of several viral oncoproteins and binds to the pocket region of Rb both in vitro and in vivo. Elf-1 binds exclusively to the underphosphorylated form of Rb and fails to bind to Rb mutants derived from patients with retinoblastoma. Co-immunoprecipitation experiments demonstrated an association between Elf-1 and Rb in resting normal human T cells. After T cell activation, the phosphorylation of Rb results in the release of Elf-1, which is correlated temporally with the activation of Elf-1-mediated transcription. Overexpression of a phosphorylation-defective form of Rb inhibited Elf-1-dependent transcription during T cell activation. These results demonstrate that Rb interacts specifically with a lineage-restricted Ets transcription factor. This regulated interaction may be important for the coordination of lineage-specific effector functions such as lymphokine production with cell cycle progression in activated T cells.

Regulation of lymphokine messenger RNA stability by a surface-mediated T cell activation pathway.

Quiescent T cells can be induced to express many genes by mitogen or antigen stimulation. The messenger RNAs of some of these genes undergo relatively rapid degradation compared to messenger RNAs from constitutively expressed genes. A T cell activation pathway that specifically regulates the stability of messenger RNAs for the lymphokines interleukin-2, interferon-gamma, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor is induced by stimulation of the CD28 surface molecule. This pathway does not directly affect the steady-state messenger RNA level, transcription, or messenger RNA half-life of other T cell activation genes, including c-myc, c-fos, IL-2 receptor, and the 4F2HC surface antigen. These data show that stimuli received at the cell surface can alter gene expression by inducing specific changes in messenger RNA degradation.

Sequence-specific binding of human Ets-1 to the T cell receptor alpha gene enhancer.

Expression of the human T cell receptor (TCR) alpha gene is regulated by a T cell-specific transcriptional enhancer that is located 4.5 kilobases (kb) 3' to the C alpha gene segment. The core enhancer contains two nuclear protein binding sites, T alpha 1 and T alpha 2, which are essential for full enhancer activity. T alpha 1 contains a consensus cyclic adenosine monophosphate (cAMP) response element (CRE) and binds a set of ubiquitously expressed CRE binding proteins. In contrast, the transcription factors that interact with the T alpha 2 site have not been defined. In this report, a lambda gt11 expression protocol was used to isolate a complementary DNA (cDNA) that programs the expression of a T alpha 2 binding protein. DNA sequence analysis demonstrated that this clone encodes the human ets-1 proto-oncogene. Lysogen extracts produced with this cDNA clone contained a beta-galactosidase-Ets-1 fusion protein that bound specifically to a synthetic T alpha 2 oligonucleotide. The Ets-1 binding site was localized to a 17-base pair (bp) region from the 3' end of T alpha 2. Mutation of five nucleotides within this sequence abolished both Ets-1 binding and the activity of the TCR alpha enhancer in T cells. These results demonstrate that Ets-1 binds in a sequence-specific fashion to the human TCR alpha enhancer and suggest that this developmentally regulated proto-oncogene functions in regulating TCR alpha gene expression.