Human T-cell Antigen Receptor Research Articles

Synthetic dual co-stimulation increases the potency of HIT and TCR-targeted cell therapies.

Chimeric antigen receptor T cells have dramatically improved the treatment of hematologic malignancies. T cell antigen receptor (TCR)-based cell therapies are yet to achieve comparable outcomes. Importantly, chimeric antigen receptors not only target selected antigens but also reprogram T cell functions through the co-stimulatory pathways that they engage upon antigen recognition. We show here that a fusion receptor comprising the CD80 ectodomain and the 4-1BB cytoplasmic domain, termed 80BB, acts as both a ligand and a receptor to engage the CD28 and 4-1BB pathways, thereby increasing the antitumor potency of human leukocyte antigen-independent TCR (HIT) receptor- or TCR-engineered T cells and tumor-infiltrating lymphocytes. Furthermore, 80BB serves as a switch receptor that provides agonistic 4-1BB co-stimulation upon its ligation by the inhibitory CTLA4 molecule. By combining multiple co-stimulatory features in a single antigen-agnostic synthetic receptor, 80BB is a promising tool to sustain CD3-dependent T cell responses in a wide range of targeted immunotherapies.

Small size of membrane-tethered ligands regulates phosphatase segregation in γδ TCR triggering

Abstract Ligand recognition by the T cell antigen receptor (TCR) is central to effective immunity. Unlike αβ T cells, γδ T cells do not recognize pMHC antigens or use coreceptors CD4/8. Instead they recognize invariant membrane-tethered ligands, which are upregulated at sites of inflammation and infection. The substantial structural similarities between αβ and γδ TCRs prompted us to investigate whether they share triggering mechanisms. However, recent studies show that binding-induced γδ TCR conformational changes are not induced by physiological ligands, leaving open the possibility that other mechanisms are involved. We investigated the stress-inducible murine surface protein T22, that is recognized by the G8 γδ TCR - since detailed biophysical and structural information is available for this system. Here we tested whether ‘size-based’ kinase-phosphatase segregation (also known as the kinetic-segregation model) mediates G8 TCR signal initiation. Elongation of the T22 ectodomain leads to failure of TCR phosphorylation and IL-2 release. Electron microscopy showed that elongated T22/G8 leads to increased intermembrane spacing at the immunological synapse. Conversely, truncation of the large CD45 ectodomain attenuated triggering, pointing to a critical dependence on ‘close contact’ for phosphatase exclusion. To establish the physiological relevance of our findings, we confirmed that T22 elongation abrogates triggering in primary G8 γδ T cells, and using a second γδ TCR (KN6). We are currently completing investigations on the role of kinetic-segregation in hapten-specific and human γδ TCR triggering. Taken together, our findings establish that γδ TCR triggering occurs primarily through the kinetic-segregation mechanism.

Detection of constant domain of human T cell antigen receptor alpha-chain via novel monoclonal antibody 7F18.

The αβ T cell antigen receptor (TCR) endows T lymphocytes with immune specificity and controls their effector functions. Each person possesses a vast repertoire of TCRs that is generated by the well-studied processes of somatic recombination and thymic selection. While many antibodies specific for TCRβ variable domains are available, antibodies specific for human TCRα are rare. We now report a novel monoclonal antibody, 7F18, which binds to human TCRα constant region, with specificity for a denatured epitope that can be visualized by SDS-PAGE followed by Western blot. Both immature and mature TCR α-chain products can be visualized, making 7F18 potentially applicable to various biochemical assays of multiprotein complex assembly and maturation. This new monoclonal antibody provides a tool that can potentially facilitate the biochemical analysis of comprehensive populations of human αβ TCR complexes that need not be limited to small subsets of the repertoire.

Butyrophilin 3A1 binds phosphorylated antigens and stimulates human γδ T cells

Human T cells that express a T cell antigen receptor (TCR) containing γ-chain variable region 9 and δ-chain variable region 2 (Vγ9Vδ2) recognize phosphorylated prenyl metabolites as antigens in the presence of antigen-presenting cells but independently of major histocompatibility complex (MHC), the MHC class I-related molecule MR1 and antigen-presenting CD1 molecules. Here we used genetic approaches to identify the molecule that binds and presents phosphorylated antigens. We found that the butyrophilin BTN3A1 bound phosphorylated antigens with low affinity, at a stoichiometry of 1:1, and stimulated mouse T cells with transgenic expression of a human Vγ9Vδ2 TCR. The structures of the BTN3A1 distal domain in complex with host- or microbe-derived phosphorylated antigens had an immunoglobulin-like fold in which the antigens bound in a shallow pocket. Soluble Vγ9Vδ2 TCR interacted specifically with BTN3A1-antigen complexes. Accordingly, BTN3A1 represents an antigen-presenting molecule required for the activation of Vγ9Vδ2 T cells.

The T cell antigen receptor: “The Hunting of the Snark”

The quest to clone the genes encoding the T cell antigen receptor (TCR) was a tale akin to Lewis Carroll's "The Hunting of the Snark". After a long and often frustrating search, back-to-back papers reporting the discovery of the genes encoding the mouse and human TCR were finally published in the March 8, 1984 edition of Nature. In this account, I outline how my laboratory hunted the human form of the Snark, and what our discovery meant to both the immunology community and me personally. Since the isolation of the TCR genes 23 years ago, more than 30,000 papers have been published on subjects as varied as central tolerance, peripheral tolerance, TCR crystal structure, and how TCR bind to peptide/MHC structures. Numerous clinical studies involving aspects of TCR biology have also been reported. Here, I briefly discuss how the knowledge generated from our discovery has been applied to basic and clinical immunology, and where it might take us in the future.

Redirection of T Cells by Delivering a Transgenic Mouse-Derived MDM2 Tumor Antigen-Specific TCR and its Humanized Derviative Is Governed by the CD8 Coreceptor and Affects Natural Human TCR Expression

Retroviral transfer of T cell antigen receptor (TCR) genes selected by circumventing tolerance to broad tumor- and leukemia-associated antigens in human leukocyte antigen (HLA)-A*0201 (A2.1) transgenic (Tg) mice allows the therapeutic reprogramming of human T lymphocytes. Using a human CD8 x A2.1/Kb mouse derived TCR specific for natural peptide-A2.1 (pA2.1) complexes comprising residues 81-88 of the human homolog of the murine double-minute 2 oncoprotein, MDM2(81-88), we found that the heterodimeric CD8 alpha beta coreceptor, but not normally expressed homodimeric CD8 alpha alpha, is required for tetramer binding and functional redirection of TCR- transduced human T cells. CD8+T cells that received a humanized derivative of the MDM2 TCR bound pA2.1 tetramers only in the presence of an anti-human-CD8 anti-body and required more peptide than wild-type (WT) MDM2 TCR+T cells to mount equivalent cytotoxicity. They were, however, sufficiently effective in recognizing malignant targets including fresh leukemia cells. Most efficient expression of transduced TCR in human T lymphocytes was governed by mouse as compared to human constant (C) alphabeta domains, as demonstrated with partially humanized and murinized TCR of primary mouse and human origin, respectively. We further observed a reciprocal relationship between the level of Tg WT mouse relative to natural human TCR expression, resulting in T cells with decreased normal human cell surface TCR. In contrast, natural human TCR display remained unaffected after delivery of the humanized MDM2 TCR. These results provide important insights into the molecular basis of TCR gene therapy of malignant disease.

Structure of a human gammadelta T-cell antigen receptor.

T-cell antigen receptors composed of gamma and delta polypeptide chains (gammadelta TCRs) can directly recognize antigens in the form of intact proteins or non-peptide compounds, unlike alphabeta TCRs, which recognize antigens bound to major histocompatibility complex molecules (MHC). About 5% of peripheral blood T cells bear gammadelta TCRs, most of which recognize non-peptide phosphorylated antigens. Here we describe the 3.1 A resolution structure of a human gammadelta TCR from a T-cell clone that is phosphoantigen-reactive. The orientation of the variable (V) and constant (C) regions of the gammadelta TCR is unique when compared with alphabeta TCRs or antibodies, and results from an unusually small angle between the Vgamma and Cgamma domains. The complementarity-determining regions (CDRs) of the V domains exhibit a chemically reasonable binding site for phosphorylated antigens, providing a possible explanation for the canonical usage of the Vgamma9 and Vdelta2 gene segments by phosphoantigen-reactive receptors. Although the gammadelta TCR V domains are similar in overall structure to those of alphabeta TCRs, gammadelta TCR C domains are markedly different. Structural differences in Cgamma and Cdelta, and in the location of the disulphide bond between them, may enable gammadelta TCRs to form different recognition/signalling complexes than alphabeta TCRs.

Effects of HIV-1 peptides on T-cell receptor variable β chain families

Superantigens (SAGs) selectively stimulate expansion and then deletion of specific T cell antigen receptor (TCR) variable beta chain (Vβ) families. We investigated six synthetically produced HIV-1-related peptides for evidence of SAG activity: three derived all or in part from the transmembrane gp41 protein and three from the genetic sequence of the tRNA binding region. The first three were chosen because they are highly immunogenic; the second three, because their genetic sequence is completely homologous to a region of the mouse mammary tumor virus, a known superantigen. We cultured peripheral blood mononuclear cells (PBMC) of HIV-negative, healthy human donors with each of these six HIV-1 peptides. Resting and blastic CD4 + and CD8 + lymphocytes were assessed pre- and post-culture using 3-color cytofluorometry and monoclonal antibodies to CD4, CD8, and 14 human TCR Vβ families. Significance testing was done using a Student t-test. Two of the HIV-1 peptides showed possible SAG activity, one from gp41 transmembrane protein, and one from tRNA binding region. Peptide JJ1, from gp41, was associated with an increased percentage of resting and blastic Vβ 5, 8, and 21 in CD4 +, but not CD8 + lymphocytes (3/3 donors, p = 0.014, p = 0.011, and p = 0.019, respectively, for blastic CD4 + lymphocytes). Peptide JJ5, from the tRNA binding region, was associated with an increased percentage of resting and blastic Vβ 5, 12, 16, and 17 in CD8 + but not CD4 + lymphocytes (4/4 donors for blastic CD8 + lymphocytes, 3/4 for resting CD8 + lymphocytes, p < 0.05 for each Vβ family, for blastic CD8 + lymphocytes). These results suggest that peptide JJ1 may have SAG activity restricted to CD4 + lymphocytes and that peptide JJ5 may have restricted cytotoxic activity, associated with CD8 + cell responsiveness. For both, the activities would lead to increased localized cytokine production and work to the advantage of the virus. These antigens might thus represent potential targets for future antiretroviral therapy.

Predominance of one T-cell antigen receptor BV haplotype in African populations.

The human T-cell antigen receptor (TCR) is the counter-receptor for the HLA/peptide complex displayed on the surface of antigen-presenting cells. It confers antigen specificity on T lymphocytes and therefore plays a central role in pathogen recognition and host response. The most frequently used form of the TCR is a heterodimer composed of variable alpha and beta chains. We investigated allele frequencies for four variable-region gene segments of the beta chain (2S1, 3S1, 8S3, and 15S1) in 146 Caucasians and 165 Africans. The results reveal significant unexpected differences between the two populations for allele frequencies, phenotypes, genotypes, and haplotypes. Among Caucasians, there are 43 phenotypes, whereas there are 31 among the Africans studied. There are 17 haplotypes in the Caucasian sample but only 10 in Africans. This loss of diversity is largely due to the high frequency of one haplotype in the African sample which represents 65% of the informative chromosomes. At least one copy of this haplotype is present in 90% of informative individuals. As a result, 29% of Africans are homozygous for the common haplotype. Less genetic diversity at TCRBV is unexpected, since Africans usually show greater genetic diversity than other ethnic groups. For example, there are approximately twice as many HLA haplotypes in Africans compared to Caucasians. Homozygosity is also unexpected because it reduces the number of TCR variants available to recognize HLA pathogen-derived peptide complexes.

Characterization of Human TCR Vβ Gene Promoter: ROLE OF THE DODECAMER MOTIF IN PROMOTER ACTIVITY

During T-lymphocyte development, the T-cell antigen receptor (TCR) gene expression is controlled by its promoter and enhancer elements and regulated in tissue- and development stage-specific manner. To uncover the promoter function and to define positive and negative regulatory elements in TCR gene promoters, the promoter activities from 13 human TCR Vbeta genes were determined by the transient transfection system and luciferase reporter assay. Although most of the TCR Vbeta gene promoters that we tested are inactive by themselves, some promoters were found to be constitutively strong. Among them, Vbeta6.7 is the strongest. 5'-Deletion and fragmentation experiments have narrowed the full promoter activity of Vbeta6.7 to a fragment of 147 base pairs immediately 5' to the transcription initiation site. A decanucleotide motif with the consensus sequence AGTGAYRTCA has been found to be conserved in most TCR Vbeta gene promoters. There are three such decamer motifs in the promoter region of Vbeta6.7, and the contribution of each such motif to the promoter activity has been examined. Further site-directed mutagenesis analyses showed that: 1) when two Ts in the decamer were mutated, the promoter activity was totally abolished; 2) when two additional nucleotides 3' to the end of decamer were mutated, the promoter activity was decreased to two-thirds of the full level; and 3) when the element with the sequence AGTGATGTCACT was inserted into other promoters, the original weak promoters become very strong. Taken together, our data suggest that the positive regulatory element in Vbeta6.7 should be considered a dodecamer rather than a decamer and that it confers strong basal transcriptional activity on TCR Vbeta genes.

Prolactin stimulates phosphorylation of the human T-cell antigen receptor complex and ZAP-70 tyrosine kinase: a potential mechanism for its immunomodulation.

Prolactin (PRL) is an immunomodulatory hormone which promotes T-cell activation and proliferation. However, the intracellular mechanisms of this action in normal lymphocytes are unknown. Because the PRL receptor (PRLR) activates several signals also activated by the T-cell antigen receptor (TCR)/CD3 complex, we evaluated whether signaling "cross-talk" occurs between these distinct receptors. Using human thymocytes, human peripheral blood lymphocytes and the rat Nb2 lymphoma T-cell, we found that PRL induced rapid phosphorylation of multiple, TCR/CD3 complex proteins, an event required for lymphocyte activation. Two of these phosphorylated proteins were identified to be CD3 epsilon and ZAP-70 tyrosine kinase, molecules essential for TCR function. Further, PRL induced tyrosyl phosphorylation of ZAP-70 in each population of T-lymphocytes tested, demonstrating for the first time that ZAP-70 is a target of PRL action. Taken together, our results suggest that the PRLR directly affects T-lymphocyte activation by means of signaling cross-talk with the TCR/CD3 complex.

Prolactin Stimulates Phosphorylation of the Human T-Cell Antigen Receptor Complex and ZAP-70 Tyrosine Kinase: A Potential Mechanism for Its Immunomodulation

Prolactin Stimulates Phosphorylation of the Human T-Cell Antigen Receptor Complex and ZAP-70 Tyrosine Kinase: A Potential Mechanism for Its Immunomodulation

Structure of the Vdelta domain of a human gammadelta T-cell antigen receptor.

Antigen recognition by T lymphocytes is mediated by cell-surface glycoproteins known as T-cell antigen receptors (TCRs). These are composed of alpha and beta, or gamma and delta, polypeptide chains with variable (V) and constant (C) regions. In contrast to alphabeta TCRs, which recognize antigen only as peptide fragments bound to molecules of the major histocompatibility complex (MHC), gammadelta TCRs appear to recognize proteins directly, without antigen processing, and to recognize MHC molecules independently of the bound peptide. Moreover, small phosphate-containing non-peptide compounds have also been identified as ligands for certain gammadelta T cells. These studies indicate that antigen recognition by gammadelta TCRs may be fundamentally different from that by alphabeta TCRs. The three-dimensional structures of several alphabeta TCRs and TCR fragments, and their complexes with peptide-MHC or superantigens, have been determined. Here we report the crystal structure of the Vdelta domain of a human gammadelta TCR at 1.9 A resolution. A comparison with antibody and alphabeta TCR V domains reveals that the framework structure of Vdelta more closely resembles that of VH than of Valpha, Vbeta or VL (where H and L refer to heavy and light chains), whereas the relative positions and conformations of its complementarity-determining regions (CDRs) share features of both Valpha and VH. These results provide the first direct evidence that gammadelta TCRs are structurally distinct from alphabeta TCRs and, together with the observation that the CDR3 length distribution of TCR delta chains is similar to that of immunoglobulin heavy chains, are consistent with functional studies suggesting that recognition of certain antigens by gammadelta TCRs may resemble antigen recognition by antibodies.

Analysis of human T-cell antigen receptor variable beta gene usage following vaccination with recombinant HBsAg.

We analyzed the TcR Vbeta gene usage before and after vaccination with the hepatitis B vaccine since changes in the TcR Vbeta gene families would be considered to provide preliminary evidence of a mechanism to prevent HBV infection. Six healthy adult volunteers received immunizations. TcR Vbeta usage, T-cell proliferation, and HLA class II alleles were examined in peripheral blood mononuclear cells (PBMC) both before and after vaccination. Furthermore, TcR Vbeta usage in postimmunization PBMC was also compared with PBMC cultured with recombinant HBsAg (rHBsAg). The level of in vitro T-cell proliferation in the presence of rHBsAg increased significantly (P < 0.01) in PBMC isolated after vaccinations. Increases in the different TcR Vbeta genes were also observed in each individual following vaccinations, regardless of the similarity in their HLA alleles. Specific HBV-related antigen-responsive T cells were induced after HB vaccination, without any common restriction for the TcR Vbeta gene families. The mechanism that helps prevent HBV infection was thus found to involve multiclonal alterations in the TcR Vbeta repertoire.

Expression and function of apoptosis antigen fas on human and mouse immunocytes

Superantigens (SAGs) selectively stimulate expansion and then deletion of specific T cell antigen receptor (TCR) variable beta chain (Vβ) families. We investigated six synthetically produced HIV-1-related peptides for evidence of SAG activity: three derived all or in part from the transmembrane gp41 protein and three from the genetic sequence of the tRNA binding region. The first three were chosen because they are highly immunogenic; the second three, because their genetic sequence is completely homologous to a region of the mouse mammary tumor virus, a known superantigen. We cultured peripheral blood mononuclear cells (PBMC) of HIV-negative, healthy human donors with each of these six HIV-1 peptides. Resting and blastic CD4+ and CD8+ lymphocytes were assessed pre- and post-culture using 3-color cytofluorometry and monoclonal antibodies to CD4, CD8, and 14 human TCR Vβ families. Significance testing was done using a Student t-test. Two of the HIV-1 peptides showed possible SAG activity, one from gp41 transmembrane protein, and one from tRNA binding region. Peptide JJ1, from gp41, was associated with an increased percentage of resting and blastic Vβ 5, 8, and 21 in CD4+, but not CD8+ lymphocytes (3/3 donors, p = 0.014, p = 0.011, and p = 0.019, respectively, for blastic CD4+ lymphocytes). Peptide JJ5, from the tRNA binding region, was associated with an increased percentage of resting and blastic Vβ 5, 12, 16, and 17 in CD8+ but not CD4+ lymphocytes (4/4 donors for blastic CD8+ lymphocytes, 3/4 for resting CD8+ lymphocytes, p < 0.05 for each Vβ family, for blastic CD8+ lymphocytes). These results suggest that peptide JJ1 may have SAG activity restricted to CD4+ lymphocytes and that peptide JJ5 may have restricted cytotoxic activity, associated with CD8+ cell responsiveness. For both, the activities would lead to increased localized cytokine production and work to the advantage of the virus. These antigens might thus represent potential targets for future antiretroviral therapy.

Preferential rearrangements of the V gamma I subgroup of the gamma-chain of the T-cell antigen receptor to J gamma 2C gamma 2 gene segments in peripheral blood lymphocyte transcripts from normal donors.

To investigate whether there are preferential VJC gene rearrangements of the gamma-chain of the human T-cell antigen receptor (TCR), we amplified and sequenced gamma-chain TCR transcripts from peripheral blood lymphocytes from adult normal donors. cDNA was synthesized from total RNA and amplified by the polymerase chain reaction (PCR) using 5' primers specific for either the V gamma I or the V gamma II subgroups of the gamma-chain of the TCR. The amplified cDNAs were then cloned and sequenced. The majority (approximately 83%) of the cDNAs employing V-I subgroup gene segments rearranged to J gamma 2 (J gamma 2.1 or J gamma 2.3) C gamma 2 gene segments. This was in contrast to the predominant rearrangement of the V gamma II subgroup (V gamma 9) to J gamma 1.2C gamma 1. The remaining 13% of the cDNAs employing V gamma I subgroup gene segments rearranged to J gamma 1.1C gamma 1 or J gamma 1.3C gamma 1. There was significant N diversity as well as imprecise joining at the VJ junction. gamma delta TCR utilizing the C gamma 1 gene segment are disulfide-linked, whereas those utilizing the C gamma 2 gene segment are non-disulfide-linked. These results demonstrate that peripheral blood gamma-chain transcripts exhibit preferential rearrangements of V gamma I subgroup gene segments to J gamma 2(2.1,2.3)C gamma 2 gene segments. By contrast, V gamma II subgroup (V gamma 9) transcripts exhibit rearrangements to J gamma 1.2C gamma 1.

Unusual organization of the human T-cell receptor beta-chain gene complex is linked to recombination hotspots.

Two rare Sfi I polymorphisms of 360 kb and 280 kb present within the human T-cell antigen receptor beta-chain gene complex were revealed by pulsed-field gel electrophoresis. They represent allelic variants of the polymorphic 330- and 300-kb Sfi I fragments previously described. The 360-kb polymorphism results from duplication of the 30-kb DNA fragment responsible for the 330/300-kb insertion/deletion-related polymorphism. The 280-kb polymorphism results from a 20-kb deletion from the 300-kb SfiI allele. The rare polymorphisms also map on either side of a Sal I site located near a recombination hotspot, suggesting that germline duplications and deletions arose from nonhomologous crossover events.

Cutaneous T-cell lymphoma: Utility of antibodies to the variable regions of the human T-cell antigen receptor

The clonotypic 90 kd Ti heterodimer of the human T-cell antigen receptor is composed of two distinct chains (alpha beta or rarely tau delta) that result from the recombination of variable (V), constant, joining, and, in the case of beta chains, additional diversity regions. The variable region expression of human cutaneous T-cell lymphoma (CTCL) was studied. Biopsy specimens from 13 patients with CTCL (7 plaque, 3 tumor stage, 3 Sézary syndrome) were examined immunohistochemically by a panel of seven commercially available monoclonal V-region antibodies. Two patients had significant anti-V-region staining. One patient with Sézary syndrome had two lesions, subjected to biopsy 4 months apart, that reacted with beta V5(a), a specificity previously documented by flow cytometry of leukemic cells. A patient with plaque-stage CTCL, negative for T-cell gene rearrangement by Southern blot, demonstrated reactivity with beta V5(c) largely limited to epidermotropic lymphocytes. Panels of V-region antibodies should be useful reagents for diagnosis and follow-up of CTCL.

Extensive conservation of alpha and beta chains of the human T-cell antigen receptor recognizing HLA-A2 and influenza A matrix peptide.

The major histocompatibility complex class I molecule HLA-A2.1 presents the influenza A virus matrix peptide 57-68 to cytotoxic T lymphocytes in all individuals with this common HLA type and is among the most thoroughly studied immune responses in humans. We have studied the T-cell receptor (TCR) heterogeneity of T cells specific for HLA-A2 and influenza A matrix peptide using the polymerase chain reaction. The usage of V alpha and V beta sequences seen on these T cells is remarkably conserved as are certain junctional sequences associated with alpha and beta chains. Furthermore, two unrelated HLA-A2 individuals have a similar pattern of TCR usage, implying that this is a predominant response in HLA-A2 populations. Analysis in one individual showed that the conserved TCR V alpha and V beta genes are minor members of the peripheral blood TCR repertoire. The sequences provide important information on the TCR necessary for the final structural analysis of this ternary complex.

Degradation of T-cell receptor chains in the endoplasmic reticulum is inhibited by inhibitors of cysteine proteases.

The endoplasmic reticulum, or an organelle closely associated with it, contains proteases that can be used to remove partially assembled or improperly folded proteins. Very little is known at present about the types of protease that degrade these proteins. The beta chain and cluster of differentiation (CD)3 delta subunit of the human T-cell antigen receptor (TCR) are degraded shortly after synthesis. In this study Chinese hamster ovary (CHO) cells transfected with either beta or delta were incubated with a panel of protease inhibitors, and the rates of degradation of the transfected proteins were followed using chain-specific enzyme-linked immunosorbent assays (ELISAs). Of the protease inhibitors tested, degradation of both chains was highly sensitive to sulfhydryl reagents and peptidyl inhibitors of cysteine proteases. Concentrations of inhibitors that produced near complete inhibition of degradation in the endoplasmic reticulum did not cause gross changes in cellular ATP levels nor did they significantly slow constitutive secretion from CHO cells. The inhibitors did not affect the ability of CHO cells to synthesize and assemble disulphide-linked TCR zeta dimers. We conclude that the protease inhibitors were not toxic to cells and did not affect the biosynthetic activity of the endoplasmic reticulum. Furthermore, they did not alter the ability of the endoplasmic reticulum to deliver its content to the Golgi apparatus. Taken together, these results suggest that the cysteine protease inhibitors slow degradation in the endoplasmic reticulum through an action on cysteine proteases. The results imply that the endoplasmic reticulum contains cysteine proteases that can be used to remove retained proteins.