Selective CD28 blockade attenuates acute and chronic rejection of murine cardiac allografts in a CTLA-4-dependent manner.

Selective blockade of CD28 is a promising therapy to inhibit pathogenic alloimmunity. However, evaluation of this approach in transplantation has been very limited. Using a novel nonactivating single-chain Fv-based reagent (α28scFv), we have investigated the role of CD28 and cytotoxic T lymphocyte antigen 4 (CTLA-4) in a murine cardiac transplant model. Blockade of CD28 for 2 weeks after engraftment promoted allograft survival, and significantly attenuated chronic rejection when combined with transient CD154-blockade or calcineurin inhibition. Graft acceptance was associated with decreased alloantibody production, increased proportion of early graft infiltration by regulatory T cells and increased expression of regulatory dendritic cell genes. Blockade of CTLA-4 during α28scFv-based treatments led to prompt rejection in all animals and inhibited expression of forkhead box P3 (Foxp3), programmed death (PD)-1 and 2,3-indoleamine dioxygenase (IDO) in the graft. These results show that CD28 signaling during the first weeks after transplant is a pivotal mediator of pathogenic alloimmunity, and that selective CD28 blockade prolongs graft acceptance by at least two immunomodulatory mechanisms. Selective CD28 inhibition while sparing CTLA-4 is thus a promising approach to inhibit pathogenic alloimmunity.

Blockade of T cell activation using a surface-linked single-chain antibody to CTLA-4 (CD152).

CTLA-4 (CD152) engagement can down-regulate T cell activation and promote the induction of immune tolerance. However, the strategy of attenuating T cell activation by engaging CTLA-4 has been limited by sharing of its natural ligands with the costimulatory protein CD28. In the present study, a CTLA-4-specific single-chain Ab (scFv) was developed and expressed on the cell surface to promote selective engagement of this regulatory molecule. Transfectants expressing anti-CTLA-4 scFv at their surface bound soluble CTLA-4 but not soluble CD28. Coexpression of anti-CTLA-4 scFv with anti-CD3epsilon and anti-CD28 scFvs on artificial APCs reduced the proliferation and IL-2 production by resting and preactivated bulk T cells as well as CD4+ and CD8+ T cell subsets. Importantly, expression of anti-CTLA-4 scFv on the same cell surface as the TCR ligand was essential for the inhibitory effects of CTLA-4-specific ligation. CTLA-4-mediated inhibition of tyrosine phosphorylation of components of the proximal TCR signaling apparatus was similarly dependent on coexpression of TCR and CTLA-4 ligands on the same surface. These findings support a predominant role for CTLA-4 function in the modification of the proximal TCR signal. Using T cells from DO11.10 and 2C TCR transgenic mice, negative regulatory effects of selective CTLA-4 ligation were also demonstrated during the stimulation of Ag-specific CD4+ and CD8+ T cells by MHC/peptide complexes. Together these studies demonstrate that selective ligation of CTLA-4 using a membrane-bound scFv results in attenuated T cell responses only when coengaged with the TCR during T cell/APC interaction and define an approach to harnessing the immunomodulatory potential of CTLA-4-specific ligation.

Fulminant spontaneous autoimmunity of the central nervous system in mice transgenic for the myelin proteolipid protein-specific T cell receptor.

Proteolipid protein (PLP)-139-151 is the dominant encephalitogenic peptide that induces experimental autoimmune encephalomyelitis (EAE) in SJL (H-2(s)) mice. To examine the contribution of T cell receptor (TCR) specificity in the induction of EAE, we generated transgenic mice expressing the rearranged TCR genes from an encephalitogenic or a nonencephalitogenic PLP-139-151/I-A(s)-specific T cell clone. Both types of transgenic lines developed spontaneous EAE, but, remarkably, the lines expressing the TCR from the nonencephalitogenic clone showed increasingly higher frequencies of disease (60-83%) in progressive SJL backcrosses and could not be propagated on the susceptible background. The T cells from the transgenic mice were not tolerized, because they responded vigorously to the antigen in vitro and mediated EAE when the mice were immunized with antigen. Besides being the only description of a TCR transgenic mice for the PLP-139-151/I-A(s) epitope, the results demonstrate that the TCR from a nonencephalitogenic PLP-specific T cell clone can induce autoimmune disease when expressed appropriately in vivo.

IL-4, IL-10, IL-13, and TGF-beta from an altered peptide ligand-specific Th2 cell clone down-regulate adoptive transfer of experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is induced in the SJL/J mouse by adoptive transfer of activated proteolipid protein peptide (PLP) 139-151-specific Th1 cells. T cells responding to altered peptide ligands (APL) of PLP, previously shown to induce Th2 differentiation and regulate disease in PLP-immunized mice, do not transfer EAE. However, the exact mechanism of disease regulation by APL-specific T cells has not been elucidated. In this report, we show that 1F1, a Th2 clone specific for an APL of PLP139-151 can prevent adoptive transfer of EAE when cocultured with PLP-encephalitogenic spleen cells (PLP-spleen). Cytokines from activated 1F1 cells were detected by hybridization of mRNA to oligonucleotide arrays (DNA chip) and by ELISA. The Th2 cytokines found to be present at the highest protein and mRNA levels were evaluated for their role in suppression of adoptive transfer of EAE from PLP-activated spleen cell cultures. Abs to individual cytokines in 1F1 PLP-spleen cocultures suggested that IL-4, IL-13, and TGF-beta played a significant role in suppressing EAE. Abs to the combination of IL-4, IL-10, IL-13, and TGF-beta completely neutralized the protective effect of 1F1. Addition of Th2 cytokines to PLP-spleen cultures showed that IL-13 and TGF-beta were each individually effective and low levels of IL-4 synergized with IL-13 to inhibit disease transfer. IL-5, IL-9, and IL-10 had little or no effect whereas GM-CSF slightly enhanced EAE. Our results demonstrate that Th2 cytokines derived from APL-specific Th2 cells can effectively down-regulate the encephalitogenic potential of PLP-spleen cells if present during their reactivation in culture.

The murine IL-13 receptor alpha 2: molecular cloning, characterization, and comparison with murine IL-13 receptor alpha 1.

Two components of a receptor complex for IL-13, the IL-4R and a low affinity IL-13-binding chain, IL-13R alpha 1, have been cloned in mice and humans. An additional high affinity binding chain for IL-13, IL-13R alpha 2, has been described in humans. We isolated a cDNA from the thymus that encodes the murine orthologue of the human IL-13R alpha 2. The predicted protein sequence of murine IL-13R alpha 2 (mIL-13R alpha 2) has 59% overall identity to human IL-13R alpha 2 and is closely related to the murine low affinity IL-13-binding subunit, IL-13R alpha 1. The genes for both mIL-13-binding chains map to the X chromosome. A specific interaction between mIL-13R alpha 2.Fc protein and IL-13 was demonstrated by surface plasmon resonance using a BIACORE instrument. Ba/F3 cells that were transfected with mIL-13R alpha 2 expressed 5000 molecules per cell and bound IL-13 with a single Kd of 0.5 to 1.2 nM. However, these cells did not proliferate in response to IL-13, and the IL-4 dose response was unaffected by high concentrations of IL-13. In contrast, the expression of mIL-13R alpha 1 by Ba/F3 cells resulted in a sensitive proliferative response to IL-13. Consistent with its lower affinity for IL-13, IL-13R alpha 1.Fc was 100-fold less effective than IL-13R alpha 2.Fc in neutralizing IL-13 in vitro. These results show that mIL-13R alpha 2 and mIL-13R alpha 1 are not functionally equivalent and predict distinct roles for each polypeptide in IL-13R complex formation and in the modulation of IL-13 signal transduction.

Transfection of TCR alpha-chains into suppressor and T helper cell hybridomas. Production of suppressor factors with predicted antigen specificity.

Conditioned medium from Ag-specific suppressor T cell hybridomas contains soluble factors (TsF) that modulate immune responses in an Ag-specific manner. We previously generated a series of TCR-alpha- and TCR-beta- expression variants from a 4-hydroxy-3-nitrophenyl acetyl (NP)-specific inducer suppressor T cell hybridoma and demonstrated that loss of TCR alpha-chain mRNA, but not TCR-beta chain mRNA, was accompanied by concomitant loss of suppressor bioactivity. Suppressor factor bioactivity was restored by expression of TCR alpha-chain cDNA, suggesting that the TCR alpha-chain plays a critical role in Ag-specific suppressor cell function. We have now transfected TCR alpha-chain from a Th cell clone specific for arsanylated peptides plus I-Ad into a TCR-alpha- derivative of an NP-specific inducer suppressor T cell hybridoma. The transfectants expressed a new hybrid TCR-alpha beta complex and produced soluble factors that suppressed azobenzenearsonate hapten (ABA) but not NP delayed-type hypersensitivity responses. These supernatants mediated suppression of the induction, but not the effector phase of the delayed-type hypersensitivity reaction. In reciprocal experiments we transfected a TCR alpha-chain from an NP-specific suppressor T cell hybridoma into a TCR-alpha- hybridoma derived from the ABA-specific Th cell hybridoma. The NP-specific TCR alpha-chain was expressed in the Th cell hybridoma, but the supernatant from this transfectant did not suppress DTH responses to either NP or ABA. However, the latter supernatants, when combined with cell lysates derived from a TCR-alpha- Ts hybridoma, specifically suppress NP DTH responses. These data are consistent with the interpretation that TCR alpha-chain imparts Ag specificity to the suppressor molecule and a second, yet undefined, component produced by the Ts hybridoma controls the immunoregulatory bioactivity.

T cell receptor alpha-chain defines the antigen specificity of antigen-specific suppressor factor but does not impart genetic restriction.

Previous studies utilizing NP (4-hydroxy, 3-nitrophenyl acetyl hapten)-specific, T suppressor hybridomas have indicated that expression of TCR-alpha, but not TCR-beta, mRNA is required for expression of Ag-specific suppressor factor bioactivity. Suppressor-effector factor has been shown to be Ag specific and I-J restricted. Although the expression of TCR-alpha mRNA was necessary for suppressor activity, the role of TCR-alpha, as it pertained to the functional properties of T cell suppressor factor (TsF), was not established. To determine which properties of TsF could be accounted for by TCR-alpha expression, TCR-alpha cDNA, derived from NP-specific, suppressor T cell (Ts) hybridomas, was transfected into recipient Ts hybridomas of a second Ag specificity. The resulting heterologous transfectants displayed NP-specific, genetically restricted TsF activity. The Ag specificity corresponded to that of the TCR-alpha donor; however, the genetic restriction was influenced by the recipient cell, implying that TCR-alpha did not control genetic restriction of the TsF. Examination of TCR-beta expression in one of the MHC-restricted transfectants indicated that the genetic restriction of TsF could not be accounted for by TCR-beta gene products. The data support the conclusion that TCR-alpha expression is not only obligate for TsF bioactivity, but that the Ag specificity of the TCR-alpha dictates the Ag specificity of the resulting suppressor factor.

T cell receptor (TCR) usage determines disease susceptibility in experimental autoimmune encephalomyelitis: studies with TCR V beta 8.2 transgenic mice.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease that can be induced in laboratory animals by immunization with the major myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP). We analyzed the role of the T cell receptor (TCR) repertoire in susceptibility to EAE induced by these two autoantigens. Autoreactive T cells induced after immunization with MBP use a limited set of TCR. In contrast, we demonstrate that T cell clones that recognize the encephalitogenic PLP epitope (PLP 139-151) use diverse TCR genes. When the TCR repertoire is limited by introduction of a novel rearranged TCR V beta 8.2 chain in transgenic SJL mice, EAE could be induced in the transgenic mice by immunization with the encephalitogenic epitopes of PLP, but not with the encephalitogenic epitope of MBP. Thus, skewing the TCR repertoire affects the susceptibility to EAE by immunization with MBP but not with PLP. These data demonstrate the biological consequences of the usage of a more diverse T cell repertoire in the development of an autoimmune disease.

T-cell receptor alpha chain plays a critical role in antigen-specific suppressor cell function.

Antigen-specific suppressor T-cell hybridomas release soluble suppressor factors (TsF) in the supernatant that modulate both in vivo delayed-type hypersensitivity and in vitro plaque-forming cell responses in an antigen-specific manner. To study the relationship between the T-cell receptor (TcR) and TsF, we developed a series of TcR alpha- or TcR beta- expression variants from suppressor T-cell hybridomas that expressed the CD3-TcR alpha/beta complex. We demonstrate that loss of TcR alpha but not TcR beta mRNA was accompanied by the concomitant loss of suppressor bioactivity. Homologous transfection of TcR alpha cDNA into a TcR alpha- beta+ clone reconstituted both CD3-TcR expression and suppressor function. Furthermore, suppressor activity from TcR beta- variants was specifically absorbed by antigen and anti-TcR alpha antibodies, but not by anti-CD3 or anti-TcR beta affinity columns. These data directly establish a role for the TcR alpha chain in suppressor T-cell function and suggest that the TcR alpha chain is part of the antigen-specific TsF molecule.

Expression of functional alpha beta T cell receptor gene rearrangements in suppressor T cell hybridomas correlates with antigen binding, but not with suppressor cell function.

We have examined the expression of TCR genes in 4-hydroxy-3-nitrophenyl-acetyl (NP)-specific Ts cell hybridomas. Each of three independently isolated hybridomas expressed in-frame TCR alpha-chain rearrangements derived from the original suppressor Ts cell. Different V alpha and J alpha gene segments were rearranged and expressed in each Ts cell line. The only TCR beta-chain expressed in these cells was derived from the BW5147 fusion partner. Expression of the BW5147 beta-chain was found to correlate with cell surface Ag binding, inasmuch as subclones derived from one of the original Ts lines expressed greatly reduced levels of beta-chain mRNA and no longer bound to NP-coupled RBC. Subclones that continued to express beta-chain mRNA did bind to NP-coupled RBC. This suggests that the Ag receptor on Ts hybridomas is a TCR-alpha beta dimer composed of a unique alpha-chain and the BW5147 beta-chain. Ag binding could be modulated by preincubation of Ts hybridoma cells with anti-TCR-alpha beta antibody, thereby supporting this conclusion. Suppressor factor activity was measured in the conditioned media of Ts subclones that differed by 250-fold in levels of beta-chain mRNA expression. No difference in suppressor factor activity was found; conditioned media from these subclones suppressed both plaque-forming cell responses and delayed-type hypersensitivity responses at approximately equivalent dilutions. Suppressor factor activity in the conditioned media of both a beta-chain negative subclone and a beta-chain positive subclone could be absorbed with an antibody that recognizes the TCR alpha-chain, but not with an antibody that recognizes the TCR beta-chain. We conclude that suppressor factor activity in the conditioned media of these Ts hybridomas is not derived from surface TCR-alpha beta receptors, although it does share TCR alpha-chain determinants.

Novel regulators of bone formation: molecular clones and activities.

Protein extracts derived from bone can initiate the process that begins with cartilage formation and ends in de novo bone formation. The critical components of this extract, termed bone morphogenetic protein (BMP), that direct cartilage and bone formation as well as the constitutive elements supplied by the animal during this process have long remained unclear. Amino acid sequence has been derived from a highly purified preparation of BMP from bovine bone. Now, human complementary DNA clones corresponding to three polypeptides present in this BMP preparation have been isolated, and expression of the recombinant human proteins have been obtained. Each of the three (BMP-1, BMP-2A, and BMP-3) appears to be independently capable of inducing the formation of cartilage in vivo. Two of the encoded proteins (BMP-2A and BMP-3) are new members of the TGF-beta supergene family, while the third, BMP-1, appears to be a novel regulatory molecule.

Clinical effectiveness of oral and parenteral rapid neuroleptization.

A random assignment, double-blind, placebo-controlled evaluation was used to compare the effectiveness of rapid parenteral injections of fluphenazine hydrochloride with that of oral doses of the same drug in 16 patients with acute psychotic illnesses. Doses of fluphenazine hydrochloride were much higher in the parenteral neuroleptization group than in the oral administration group during the acute phase, although oral doses were equivalent during the continuation phase. The rate of improvement was not different for the two groups, but the rate of extrapyramidal side effects was higher in the parenteral neuroleptization group. The results suggest that rapid parenteral neuroleptization for the acute treatment of psychosis offers more risks than benefits.

Cortical atrophy in young adults with a history of hyperactivity in childhood.

A computed tomographic (CT) brain scan study was conducted in 24 young males treated and followed up for hyperactivity since childhood. Compared to 27 matched controls, adults with a history of hyperactivity had a significantly greater frequency of cerebral atrophy. No differences in cerebellar atrophy frequency or in lateral cerebral ventricle-to-brain ratio (VBR) were found. The possible associations of hyperactivity or perhaps stimulant drug treatment to atrophic brain changes are discussed.

Pharmacologic probes of neurotransmitter systems in tardive dyskinesia: implications for clinical management.

Despite the plethora of clinical drug trials in tardive dyskinesia, few consistent findings have emerged. One possible reason for this is that there have been no serious attempts to define the role of major neurotransmitter systems (dopamine, norepinephrine, acetylcholine, serotonin, GABA) in one specific population of tardive dyskinesia patients. This study reports a series of five controlled drug trials in a population of patients with persistent tardive dyskinesia; each drug probed one of four neurotransmitter systems. The intra- and interpatient responses are analyzed and the implications of the pharmacologic response profiles for the clinical management of tardive dyskinesia are discussed.

Cerebral ventricular enlargement in schizophrenia. A preliminary follow-up study.

Lateral cerebral ventricular enlargement is now known to occur in some schizophrenic patients. To determine whether ventriculomegaly in schizophrenia is a static vs progressive process, we conducted a follow-up computed tomographic brain scan study on 11 young male patients, three years after initial scans were obtained. No significant change was found in the mean ventricles-brain ratio of this small schizophrenic sample after three years. Four of 11 patients showed noticeable increases (greater than 50%) in individual ratio. Methodologic problems are discussed and the need for follow-up studies as a research strategy is emphasized.

Third ventricular enlargement on CT scans in schizophrenia: association with cerebellar atrophy.

The width, length, and ventricle-to-brain area ratio (VBR) of the third ventricle were measured in 55 consecutive young male schizophrenic patients and 27 matched control subjects. No differences in third ventricular dimensions were found between the two groups. However, schizophrenic patients with cerebellar atrophy had a significantly greater mean third ventricular length. Correlations of third ventricular VBR with lateral ventricular VBR, but not with sulcal widening, were found. The possible existence of a subset of schizophrenic patients defined by cerebellar atrophy and third ventricular enlargement is discussed.