Development of an Automatic Measurement Method for CD8 and PD-1 Positive T Cells Using Image Analysis Software.

BACKGROUND/AIM: With the progress in cancer immunotherapy using immune checkpoint blockade (ICB) therapy, histological observations of tumor-infiltrating lymphocyte (TIL) status are needed to evaluate the antitumor effect of ICB using imaging analysis software. MATERIALS AND METHODS: Formalin-fixed paraffin-embedded sections obtained from colorectal cancer and gastric cancer patients with more than 500 single nucleotide variants were stained with anti-CD8 and anti-PD-1 antibodies. Based on our own algorithm and imaging analysis software, an automatic TIL measurement method was established and compared to the manual counting methods. RESULTS: In the CD8+ T cell number measurement, there was a good correlation (r=0.738 by Pearson test) between the manual and automated counting methods. However, in the PD-1+ T cell measurement, there was a large difference in TIL numbers in both groups. After adjustment of the parameter settings, the correlation between the manual and automated methods in the PD-1+ T cell measurements improved (r=0.668 by Pearson test). CONCLUSION: An imaging software-based automatic measurement could be a simple and useful tool for evaluating the therapeutic effect of cancer immunotherapies in terms of TIL status.

Positron emission tomography imaging with 89Zr-labeled anti-CD8 cys-diabody reveals CD8+ cell infiltration during oncolytic virus therapy in a glioma murine model.

Determination of treatment response to immunotherapy in glioblastoma multiforme (GBM) is a process which can take months. Detection of CD8+ T cell recruitment to the tumor with a noninvasive imaging modality such as positron emission tomography (PET) may allow for tumor characterization and early evaluation of therapeutic response to immunotherapy. In this study, we utilized 89Zr-labeled anti-CD8 cys-diabody-PET to provide proof-of-concept to detect CD8+ T cell immune response to oncolytic herpes simplex virus (oHSV) M002 immunotherapy in a syngeneic GBM model. Immunocompetent mice (n = 16) were implanted intracranially with GSC005 GBM tumors, and treated with intratumoral injection of oHSV M002 or saline control. An additional non-tumor bearing cohort (n = 4) receiving oHSV M002 treatment was also evaluated. Mice were injected with 89Zr-labeled anti-CD8 cys-diabody seven days post oHSV administration and imaged with a preclinical PET scanner. Standardized uptake value (SUV) was quantified. Ex vivo tissue analyses included autoradiography and immunohistochemistry. PET imaging showed significantly higher SUV in tumors which had been treated with M002 compared to those without M002 treatment (p = 0.0207) and the non-tumor bearing M002 treated group (p = 0.0021). Accumulation in target areas, especially the spleen, was significantly reduced by blocking with the non-labeled diabody (p < 0.001). Radioactive probe accumulation in brains was consistent with CD8+ cell trafficking patterns after oHSV treatment. This PET imaging strategy could aid in distinguishing responders from non-responders during immunotherapy of GBM.

Development of a Cell Marker ELISA for the Detection of Goose T Cell Surface CD8α Molecules.

CD8 molecule is a key marker on T cell surface and is connected with the antigen recognition and activation of T lymphocytes. In order to provide a detection method for quantifying goose CD8α expression, this study raised the protein and antibody for goose CD8α and developed a feasible cell marker enzyme-linked immunoabsorbent assay (ELISA) method. Recombinant protein of the extracellular region gene of goCD8α was expressed in prokaryotic expression system, and specific polyclonal antibodies for goCD8α were raised and purified, which was further confirmed by Western-blot, immunofluorescence assay (IFA), and immunohistochemistry (IHC). A cell marker ELISA was established and optimized to detect the change of goCD8α expression between goose parvovirus (GPV)-infected and mock-infected goose peripheral blood mononuclear cells (PBMCs), which is consistent with our previously results of real-time quantitative PCR (qPCR). Cell marker ELISA can provide a new method to detect goCD8α in protein level and in a sensitive, specific, and simple way. This may provide a convenient and novel method for the detection of goCD8α expression.

Identification of a bone marrow-derived CD8αα+ dendritic cell-like population in inflamed autoimmune target tissue with capability of inducing T cell apoptosis.

DCs play critical roles in promotion of autoimmunity or immune tolerance as potent APCs. In our anti-GBM GN model, WKY rats develop severe T cell-mediated glomerular inflammation followed by fibrosis. A DC-like cell population (CD8αα(+)CD11c(+)MHC-II(+)ED1(-)) was identified in the inflamed glomeruli. Chimera experiments demonstrated that the CD8αα(+) cells were derived from BM. The CD8αα(+) cells infiltrated glomeruli at a late stage (Days 28-35), coincident with a rapid decline in glomerular inflammation before fibrosis. The CD8αα(+) cells isolated from inflamed glomeruli were able to migrate rapidly from the bloodstream into inflamed glomeruli but not into normal glomeruli, suggesting that the migration was triggered by local inflammation. Despite high-level expression of surface and cellular MHC class II molecules, in vitro experiments showed that this CD8αα(+) DC-like cell induced apoptosis but not proliferation in antigen-specific CD4(+) T cells from T cell lines or freshly isolated from lymph nodes; they were not able to do so in the absence of antigens, suggesting induction of apoptosis was antigen-specific. Furthermore, apoptotic T cells were detected in a large number in the glomeruli at Day 32, coincident with the infiltration of the cells into glomeruli, suggesting that the cells may also induce T cell apoptosis in vivo. A potential role of this CD8αα(+) DC-like population in peripheral immune tolerance and/or termination of autoimmune inflammation was discussed.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of rhesus macaque CD8alphaalpha homodimer.

As a T-cell co-receptor, CD8 binds to MHC class I molecules and plays a pivotal role in the activation of cytotoxic T lymphocytes. To date, structures of CD8 have been solved for two different mammals: human and mouse. The infection of rhesus macaques (Macaca mulatta) by simian immunodeficiency virus (SIV) is the best animal model for studying HIV. In this study, the rhesus macaque CD8 (rCD8) alphaalpha homodimer was obtained and rCD8alpha exodomain protein crystals were successfully obtained for further structural analysis. Diffraction data were collected to a resolution of 2.4 A. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 46.52, b = 56.28, c = 82.40 A. These data will facilitate further studies on the structural differences between these CD8 structures and the cellular immune responses of rhesus macaque.

Rapid immunosorting of transmembrane proteins of Lymphocytes from a cDNA expression library of COS-1 cells.

The proteins on lymphocyte surface play important roles in a wide range of immunological processes, but the profile and characterization of surface proteins remain to be further investigated, among which the method for fast screening of surface proteins needs to be established. In this study, a conventional cDNA clone library of hepatic lymphocytes from C57BL/6 mouse was constructed, and then the cDNA was inserted into a recombinant expression vector pSecTag-attR with a signal peptide and tag protein for fluorescence screening. The recombinant cDNA expression library was transfected into COS-1 cells, and the transfected cells with the expressed membrane proteins were labeled by fluorescence antibodies and isolated by fluorescence activated cell sorting. After two cycles of sorting, the purity of fluorescence positive cells with membrane proteins was up to 98%, and the representative membrane molecules on lymphocytes such as CD3, CD4, CD8, NK1.1 and NKG2D were detected in the library. These results demonstrated that the cDNA expression library containing transmembrane proteins provided an efficient and fast tool for the study of transmembrane proteins on hepatic lymphocytes.

CD8 alpha is expressed by human monocytes and enhances Fc gamma R-dependent responses.

BACKGROUND: CD8 alpha enhances the responses of antigen-specific CTL activated through TCR through binding MHC class I, favoring lipid raft partitioning of TCR, and inducing intracellular signaling. CD8 alpha is also found on dendritic cells and rat macrophages, but whether CD8 alpha enhances responses of a partner receptor, like TCR, to activate these cells is not known. TCR and FcR, use analogous or occasionally interchangeable signaling mechanisms suggesting the possibility that CD8 alpha co-activates FcR responses. Interestingly, CD8 alpha+ monocytes are often associated with rat models of disease involving immune-complex deposition and FcR-mediated pathology, such as arthritis, glomerulonephritis, ischaemia, and tumors. While rat macrophages have been shown to express CD8 alpha evidence for CD8 alpha expression by mouse or human monocytes or macrophages was incomplete. RESULTS: We detected CD8 alpha, but not CD8 beta on human monocytes and the monocytic cell line THP-1 by flow cytometry. Reactivity of anti-CD8 alpha mAb with monocytes is at least partly independent of FcR as anti-CD8 alpha mAb detect CD8 alpha by western blot and inhibit binding of MHC class I tetramers. CD8 alpha mRNA is also found in monocytes and THP-1 suggesting CD8 alpha is synthesized by monocytes and not acquired from other CD8 alpha+ cell types. Interestingly, CD8 alpha from monocytes and blood T cells presented distinguishable patterns by 2-D electrophoresis. Anti-CD8 alpha mAb alone did not activate monocyte TNF release. In comparison, TNF release by human monocytes stimulated in a FcR-dependent manner with immune-complexes was enhanced by inclusion of anti-CD8 alpha mAb in immune-complexes. CONCLUSION: Human monocytes express CD8 alpha. Co-engagement of CD8 alpha and FcR enhances monocyte TNF release, suggesting FcR may be a novel partner receptor for CD8 alpha on innate immune cells.

Expansion of human CMV-specific cytotoxic T lymphocytes to a clinical scale: a simple culture system using tetrameric HLA-peptide complexes.

BACKGROUND: Recipients of allogeneic stem cell transplants (SCT) are at risk of human CMV infection during their immunocompromised period. The increasing number of reports of CMV isolates resistant to ganciclovir after transplantation has led us to attempt to develop alternative strategies for preventing or treating CMV infection. This study describes a system for generating sufficient numbers of CMV-specific cytotoxic T lymphocytes (CTL) for adoptive immunotherapy after SCT. METHODS: CMV-specific CTL were isolated from a single blood draw of a CMV-seropositive donor using PE-labeled HLA-A*0201/pp65(495-503) tetramers and anti-PE magnetic beads. A mixture of a tetramer-positive population and CD4(+) T lymphocytes was expanded to sufficient numbers for clinical application with IL-2 and immobilized anti-CD3 stimulation. RESULT: Starting from 50 mL of blood, we generated >10(7)/m(2) tetramer-positive CTL within 2 weeks. Flow cytometric analysis of expanded lymphocytes showed that purity of CMV peptide-specific CTL was >75%. Upon stimulation of HLA-A*0201-restricted CMV peptide, expanded CD8 T lymphocytes produced intracellular IFN-gamma. Purified CTL exhibited cytotoxic activity against CMV peptide-pulsed T2 cells and CMV-infected HLA-A*0201-positive fibroblasts, but not against HLA mismatched or uninfected target cells. Alloreactivity could be excluded in MLC. DISCUSSION: This simple, rapid culture system can be useful for adoptive immunotherapy after allogeneic SCT. We are now trying to adapt our laboratory scale study to a clinical scale study under good manufacturing practices (GMP) conditions.

HLA-DR expression on lymphocyte subsets as a marker of disease activity in patients with systemic lupus erythematosus.

A major problem in the management of SLE patients is to predict a flare or to distinguish between active and quiescent disease. Serological markers are widely used to assess disease activity, but many patients have close to or normal values for these parameters while exhibiting obvious disease-related signs and symptoms. This study aimed to determine which serological parameters, among ESR, ANA and anti-dsDNA antibody titres, CH50 and the HLA-DR expression on circulating T-lymphocyte subsets, best reflected the development of SLE flares. Sixty SLE patients were included, 34 with quiescent disease throughout the entire follow-up period and 26 who experienced an SLE flare defined as having active disease. According to univariate analysis, all parameters were significantly higher for patients with active disease, with the percentage of CD8+DR+ cells being the most significant parameter (P = 10-7). Multivariate logistic regression analysis identified three independent variables enabling the identification of a lupus flare: CH50, the CD8+DR+ and CD4+DR+ cell percentages among total lymphocytes. The CD8+DR+ cell percentage is the biological parameter most significantly associated with a flare (P < 0.001), even more powerful than CH50 (P < 0.01). HLA-DR expression on CD8+ lymphocytes clearly coincided with disease evolution in seven patients enrolled as having quiescent disease, but who experienced one flare during follow-up that subsequently resolved. The percentage of circulating CD8+DR+ lymphocytes appears to be a biological marker which accurately reflects disease activity. A larger prospective study is needed to demonstrate the real efficacy of this marker in predicting an exacerbation in SLE patients.

Expression and purification of antigenically active soluble derivatives of the heterodimeric and homodimeric forms of the mouse CD8 lymphocyte membrane glycoprotein.

The T lymphocyte membrane glycoprotein CD8 enhances antigen recognition by class I-restricted T cells. There are two naturally occurring forms of CD8, an alphabeta heterodimer expressed by the majority of CD8(+) T cells, and a less abundant alphaalpha homodimer found on specialised T cell subsets. An expression strategy was developed for production of soluble CD8alphaalpha and CD8alphabeta extracellular domains for use in ligand binding studies. Mouse CD8alpha was expressed autonomously as a homodimer at 10 mg/l in mammalian fibroblasts, but CD8beta was not expressed at significant levels in the absence of CD8alpha. Co-expression with CD8alpha led to significant enhancement in the level of CD8beta expression, which was secreted as a non-covalent heterodimer at 3 mg/l with CD8alpha. Despite the marked increase of CD8beta expression in the presence of CD8alpha, an excess of soluble CD8alphaalpha homodimer was also present in the supernatant of co-expressing cell clones. In order to resolve the CD8alphaalpha homodimer from the CD8alphabeta heterodimer, affinity chromatographic techniques specific for the CD8beta subunit were employed. Purification procedures requiring elution from affinity matrices at low pH led to substantial losses in the total antigenic activity and partial subunit dissociation of the soluble CD8alphabeta heterodimer. The inclusion of a hexahistidine tag at the C-terminus of CD8beta enabled affinity purification of soluble CD8alphabeta (and sCD8alphaalpha) under neutral conditions, yielding recombinant protein with the correct stoichiometry and full antigenic activity. This method may prove useful for production of other soluble recombinant heterodimeric receptor proteins whose antigenicity is affected by denaturation during immunoaffinity purification.

Description of an ectothermic TCR coreceptor, CD8 alpha, in rainbow trout.

We have cloned the first CD8 alpha gene from an ectothermic source using a degenerate primer for Ig superfamily V domains. Similar to homologues in higher vertebrates, the rainbow trout CD8 alpha gene encodes a 204-aa mature protein composed of two extracellular domains including an Ig superfamily V domain and hinge region. Differing from mammalian CD8 alpha V domains, lower vertebrate (trout and chicken) sequences do not contain the extra cysteine residue (C strand) involved in the abnormal intrachain disulfide bridging within the CD8 alpha V domain of mice and rats. The trout membrane proximal hinge region contains the two essential cysteine residues involved in CD8 dimerization (alpha alpha or alpha beta) and threonine, serine, and proline residues which may be involved in multiple O-linked glycosylation events. Although the transmembrane region is well conserved in all CD8 alpha sequences analyzed to date, the putative trout cytoplasmic region differs and, in fact, lacks the consensus p56lck motif common to other CD8 alpha sequences. We then determined that the trout CD8 alpha genomic structure is similar to that of humans (six exons) but differs from that of mice (five exons). Additionally, Northern blotting and RT-PCR demonstrate that trout CD8 alpha is expressed at high levels within the thymus and at weaker levels in the spleen, kidney, intestine, and peripheral blood leukocytes. Finally, we show that trout CD8 alpha can be expressed on the surface of cells via transfection. Together, our results demonstrate that the basic structure and expression of CD8 alpha has been maintained for more than 400 million years of evolution.

Mucosal mast cell responses are not required for protection against infection with the murine nematode parasite Trichuris muris.

The involvement of mucosal mast cells (MMC) in protection against infection with the murine nematode parasite Trichuris muris was studied in genetically mast cell-deficient WBB6F1-W/Wv mice and their normal littermates WBB6F1-+/+ mice. Expulsion of T. muris worms occurred in infected +/+ mice, whereas no worm expulsion was observed in infected W/Wv mice where the infection persisted until at least day 46 postinfection. No MMC responses were induced in either infected W/Wv or +/+ mice. Specific IgG1and IgG2a antibodies to T. muris excretory/secretory antigens were observed in infected W/Wv and +/+ mice, and antibody production showed similar kinetics. Interleukin 4 production by concanavalin A (Con A)-stimulated mesenteric lymph node cells (MLNC) was induced preferentially in infected +/+ mice. T. muris infection increased the levels of IFN-gamma produced by Con A-stimulated MLNC of infected W/Wv and +/+ mice, with the levels of IFN-gamma in infected W/Wv mice being higher than those in infected +/+ mice. Taken together, these results indicate that W/Wv and +/+ mice are susceptible and resistant to T. muris infection, respectively, and that MMC responses are not required for protective immunity.

A model for the origin of TCR-alphabeta+ CD4-CD8- B220+ cells based on high affinity TCR signals.

The origin of TCR-alphabeta+ CD4-CD8- cells is unclear, yet accumulating evidence suggests that they do not represent merely a default pathway of unselected thymocytes. Rather, they arise by active selection as evidenced by their absence in mice lacking expression of class I MHC. TCR-alphabeta+ CD4-CD8- cells also preferentially accumulate in mice lacking expression of Fas/APO-1/CD95 (lpr) or Fas-ligand (gld), suggesting that this subset might represent a subpopulation destined for apoptosis in normal mice. Findings from mice bearing a self-reactive TCR transgene support this view. In the current study we observe that in normal mice, TCR-alphabeta+ CD4-CD8- thymocytes contain a high proportion of cells undergoing apoptosis. The apoptotic subpopulation is further identified by its expression of B220 and IL2Rbeta and the absence of surface CD2. The CD4-CD8- B220+ phenotype is also enriched in T cells that recognize endogenous retroviral superantigens, and can be induced in TCR transgenic mice using peptide/MHC complexes that bear high affinity, but not low affinity, for TCR. A model is presented whereby the TCR-alphabeta+ CD2- CD4-CD8- B220+ phenotype arises from high intensity TCR signals. This model is broadly applicable to developing thymocytes as well as mature peripheral T cells and may represent the phenotype of self-reactive T cells that are increased in certain autoimmune conditions.

Regulation of apoptosis in mature alphabeta+CD4-CD8- antigen-specific suppressor T cell clones.

The regulation of apoptosis in mature CD4+ or CD8+ alphabeta+ T cells has been well studied. How the survival and death is regulated in peripheral CD4-CD8- (double negative, DN) alphabeta+ T cells remains unknown. Recent studies suggest that peripheral DN T cells may play an important role in the regulation of the immune responses mediated by CD4+ or CD8+ T cells. Here, we used immunosuppressive DN T cell clones to elucidate the mechanisms involved in the regulation of death and survival of alphabeta+ DN T cells. The DN T cell clones were generated from the spleen cells of 2C transgenic mice, which express the transgenic TCR specific for Ld and permanently accepted Ld+ skin allografts after pretransplant infusion of Ld+ lymphocytes. We report that 1) the mature DN T cells are highly resistant to TCR cross-linking-induced apoptosis in the presence of exogenous IL-4; 2) Fas/Fas-ligand and TNF-alpha/TNFR pathways do not play an apparent role in regulating apoptosis in DN T cells; 3) the DN T cells constitutively express a high level of Bcl-xL, but not Bcl-2; 4) both Bcl-xL and Bcl-2 are up-regulated following TCR-cross-linking; and 5) IL-4 stimulation significantly up-regulates Bcl-xL and c-Jun expression and leads to mitogen-activated protein kinase phosphorylation in DN T cells, which may contribute to the resistance to apoptosis in these T cells. Taken together, these results provide us with an insight into how mature DN T cells resist activation-induced apoptosis to provide a long-term suppressor function in vivo.

Role of peripheral blood mononuclear cell (PBMC) phenotype changes in the pathogenesis of haemorrhagic fever with renal syndrome (HFRS).

Hantaviruses cause an important human illness, HFRS. Blood samples from 22 HFRS-positive, six seronegative patients and 15 healthy controls were examined in 1995, during the largest HFRS epidemic in Croatia. Results of double- and triple-colour immunofluorescence analysis showed an increased percentage of cytotoxic T cells (CD3+CD8+) in seropositive patients compared with seronegatives and healthy controls. The majority of seropositive HFRS patients expressed activation and memory antigens on T and B lymphocytes. The percentage of CD23+ and CD21+ B lymphocytes was lower in seropositive patients. HFRS patients had elevated levels of sCD23 and five had elevated total IgE. The increased expression of both early and late T cell activation antigens, e.g. CD25, CD71 and HLA-DR, memory cells and sCD23 positively correlated with biochemical parameters (AST, ALT, urea, alpha2-globulin) during the acute phase of HFRS. The phenotypic changes observed, especially early and late T cell activation markers, as well as memory cells, could be useful parameters in the evaluation of HFRS course, and prognostic factors of HFRS severity. Additional attention should be paid to liver involvement in the pathogenesis of HFRS.

Circulating TCR gammadelta cells in the patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a disorder with a wide range of immunological abnormalities. The results of the studies undertaken in the last decade indicated that SLE pathogenesis was mainly connected with the breakdown of the activation control of B and T cells, generating humoral or cell-mediated responses against several self-antigens of affected cells. The last studies demonstrate that the role of gammadelta T lymphocytes in autoimmune diseases can be especially important. Flow cytometry techniques were used to investigate the number and percentage of TCR gammadelta T cells and their most frequent subtypes in peripheral blood of 32 patients with SLE and 16 healthy volunteers. We also correlated TCR gammadelta cells number with the level of T CD3+, T CD4+, T CD8+, and NK (CD16) cells (cytometric measurements) and SLE activity (on the basis of clinical investigations). Our studies were preliminary attempts to evaluate the role of that minor T cell subpopulation in SLE. Absolute numbers of cells expressing gammadelta TCR in most SLE blood specimens were significantly lower than in the control group (P<0.006). However, since the level of total T cell population was also decreased in the case of SLE, the mean values of the percentage gammadelta T cells of pan T lymphocytes were almost the same in both analysed populations (7.1% vs 6.3%, respectively). In contrast to Vdelta2+ and Vgamma9+ subtypes of pan gammadelta T cells, Vdelta3+ T cells number was higher in SLE patients (20 x 10 cells/microl) than in healthy control group (2 x 2 cells/microl) (P=0.001). However, we found no differences between the numbers of pan gammadelta T lymphocytes and studied their subtypes in the patients with active and inactive disease. These cell subpopulations were doubled in the treated patients with immunosuppressive agents in comparison with untreated ones; however, data were not statistically significant. Our study indicated that Vdelta3+ subtype of gammadelta T cells seems to be involved in SLE pathogenesis; however, we accept the idea that the autoimmunity does not develop from a single abnormality, but rather from a number of different events.

CD8 dimer usage on alpha beta and gama delta T lymphocytes from equine lymphoid tissues.

Eight murine monoclonal antibodies (mAb) were used to identify the equine CD8 alpha or CD8 beta chains and to define the expression of these chains on lymphocytes from various lymphoid tissues. CD8 alpha was a 39 kDa protein and CD8 beta was a 32 kDa protein. Both chains were expressed on most of the CD8+ T lymphocytes in the peripheral blood, spleen, thymus, mesenteric lymph nodes and ileal intraepithelial lymphocytes (IEL), however, in each lymphoid compartment a percentage of lymphocytes expressed only the CD8 alpha chain. The largest percentage of CD8 alpha alpha expressing T lymphocytes was 37.7% of the IELs. Purified T lymphocytes from the ileum expressing CD8 alpha beta co-expressed the alpha beta T cell receptor (TCR). In contrast, purified CD8+ T lymphocytes from the PBMC co-expressed either the alpha beta or gamma delta TCR by RT-PCR. Use of pooled anti-CD8 alpha mAb of the murine IgG2a isotype and rabbit complement resulted in lysis of the entire CD8 expressing population in peripheral blood mononuclear cells (PBMC). These results indicated that CD8 dimer usage by equine T lymphocytes is similar to other species and that the mAb described can be further used to separate equine CD8+ T lymphocyte subsets from the lymphoid tissues to define their function in protection against viral and other infections.

Changes in arrangement and in conformation of molecular components of peripheral T-cell antigen receptor complex after ligand binding: analyses by co-precipitation profiles.

Amounts of co-precipitating CD3 components by anti-T-cell receptor (TCR)V beta or anti-CD4/8 monoclonal antibodies were compared between non-stimulated and stimulated splenic T cells. The amounts of co-precipitating CD3 delta, epsilon and gamma chains with TCR alpha beta and with CD4/8 were not significantly changed after TCR ligation. The apparent amount of CD3 zeta chain co-precipitated with TCR alpha beta increased up to threefold, while the actual amount of co-precipitating CD3 zeta with TCR alpha beta and the total amount of specifically precipitated CD3 zeta are not changed after cross linking of TCR. The apparent amount of CD3 zeta chain co-precipitated with CD4/8 also increased. Unlike co-precipitation with TCR alpha beta, the actual amount of CD3 zeta co-precipitated with CD4/8 increased significantly. This observation suggests a conformational change as well as the relocation of CD3 zeta molecules within the TCR complex after the signal delivery. After TCR ligation, CD3 zeta chains relocate to the vicinity of either CD4 or CD8 molecules. In addition, when cross linking and binding signals are compared, CD3 chains undergo two distinct phases of conformational change. The responses, while the later conformational change caused by the cross linking of TCR does not induce but enhances the proliferative response.

Production of crystallizable fragments of membrane proteins.

Many membrane proteins feature autonomously folded extramembranous domains which, when isolated from the intact protein, perform biochemical functions relevant to biological activity. Whereas intact membrane proteins usually require detergent solubilization for purification, most extramembranous fragments are soluble in aqueous solution. If appropriately constructed, such fragments are often crystallizable and the resulting atomic structures can lead to important biological insight. In most instances, these fragments are produced in recombinant expression systems. To be crystallizable, molecular fragments should be uniform in composition and conformation and be available in abundance. Considerations for the production of crystallizable fragments of membrane proteins include the definition of fragment boundaries, the control of nonuniformities introduced by glycosylation of phosphorylation, and optimization of expression systems. These aspects are addressed here in general terms and in the case studies of applications to CD4, CD8, the insulin receptor kinase, and N-cadherin.