Analysis of regulatory phosphorylation sites in ZAP-70 by capillary high-performance liquid chromatography coupled to electrospray ionization or matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

A methodology for the rapid and quantitative analysis of phosphorylation sites in proteins is presented. The coupling of capillary high-performance liquid chromatography (HPLC) to electrospray ionization mass spectrometry (ESI-MS) allowed one to distinguish phosphorylation sites based on retention time and mass difference from complex peptide mixtures. The methodology was first evaluated and validated for a mixture of non-, mono-, and dityrosine-phosphorylated synthetic peptides, corresponding to the tryptic fragment 485-496 (ALGADDSYYTAR) of the human protein tyrosine kinase ZAP-70. The limits of detection for the non-, mono- and diphosphorylated peptides were about 15, 40 and 100 fmol, respectively, when using a 300 microm I.D. column. Application of the method was extended to identify phosphopeptides generated from a trypsin digest of recombinant autophosphorylated ZAP-70, in particular with respect to quantifying the status at the regulatory phosphorylation sites Tyr-492 and Tyr-493. Combination of chromatographic and on-line tandem mass spectrometry data allowed one to ascertain the identity of the detected peptides, a prerequisite to analyses in more complex biological samples. As an extension to the methodology described above, we evaluated the feasibility of interfacing capillary HPLC to matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), using a micromachined piezoelectric flow-through dispenser as the interface. This enabled direct arraying of chromatographically separated components onto a target plate that was precoated with matrix for subsequent analysis by MALDI-TOF-MS without further sample handling.

Differential regulation of p72syk expression in naive and proliferating CD4-CD8+ T cells.

A well-known consequence of TCR stimulation in proliferating T cells is cell death by apoptosis. We have previously shown that the extent of tyrosine phosphorylation of TCR zeta, CD3 gamma, and CD3 epsilon subunits in proliferating CD4-CD8+ T cells after TCR stimulation was decreased when compared to similarly stimulated naive T cells expressing the same TCR. Furthermore, these differences correlated with a decrease in the specific kinase activity of p56lck and p59fyn, with a corresponding increase in the specific kinase activity of p50rsk, a negative regulator of src-family tyrosine kinases. In this study we determined whether kinases that bind tyrosine phosphorylated TCR zeta chain were differentially regulated in naive and proliferating cells. Chemically synthesized cytoplasmic domains of the TCR zeta chain were fully phosphorylated in vitro with p56lck and used to precipitate TCR zeta binding proteins in naive and proliferating cells. Using this method we found that both ZAP-70 and p72syk bound tyrosine phosphorylated TCR zeta very efficiently. More interestingly, p72syk was found to be expressed only in naive but not proliferating cells. Kinetic studies indicate that more than 48 hr of activation was required for ceasation of p72syk expression. We also showed that the inability to detect p72syk expression in proliferating cells was not due to its translocation to cytoskeletal compartments in proliferating cells. We propose that the differential regulation of ZAP-70 and p72syk in naive and proliferating cells may contribute to the uncoupling of the TCR signaling pathway from downstream signaling events leading to distinct functional outcomes in these two cell types after TCR stimulation.

Differential activation of phospholipase C-gamma 1 and mitogen-activated protein kinase in naive and antigen-primed CD4 T cells by the peptide/MHC ligand.

In this study, we determined the functional and biochemical differences in naive and primed CD4 T cells that expressed a TCR specific for the pigeon cytochrome c (pcc) peptide presented by I-Ek MHC class II molecules. Naive CD4 T cells expressing the transgenic TCR were isolated from the peripheral lymphoid organs of transgenic mice and stimulated with pcc peptide and IL-2 for 10 to 14 days. After this culture period, the Ag-primed cells were quiescent, as judged by the lack of expression of the early activation marker CD69, low expression of CD25 (IL-2R), and failure to incorporate thymidine. The primed cells required 10-fold less peptide than naive cells to achieve the same degree of proliferation and for the induction of CD69. Primed cells also mobilized calcium more efficiently with regard to Ag dose and magnitude of the response. The biochemical signal-transduction events in naive and primed T cells were compared by stimulating them with different concentrations of pcc peptide presented by adherent Ek-transfected fibroblasts. It was found that tyrosine phosphorylation and activation of mitogen-activated protein kinase (MAPK) in primed cells required 10-fold less Ag and occurred more rapidly and intensively. Interestingly, peptide stimulation induced tyrosine phosphorylation of phospholipase C (PLC)-gamma 1 exclusively in primed cells. RasGAP was also more efficiently tyrosine phosphorylated in primed cells. By contrast, Shc was tyrosine phosphorylated to the same extent in naive and primed cells. PI3Kp85 was not tyrosine-phosphorylated in naive and primed cells either before or after peptide stimulation. We propose that the higher sensitivity of the primed cells to Ag stimulation is most likely dependent, at last in part, on the more efficient activation of PLC-gamma 1, MAPK, and calcium-dependent pathways.

Expression of CD11a and CD45R isoforms defines distinct subsets of CD8+ TCR alpha beta and TCR gamma delta CTL in vivo.

Intraperitoneal immunization of rats with a syngeneic lymphoma and allogeneic leukocytes induced enrichment of antigen-selective TCR alpha beta+ and TCR gamma delta+ CTL. The peritoneal cavity seems to be a suitable site for enrichment of antigen-selective CTL, since the induced effector cells executed strong cytotoxicity without any requirement for in vitro reactivation. Tumor-selective CTL expressed high cell surface levels of CD45RC, allogeneic CTL expressed a variable level of CD45RC, while SAg-reactive CTL had low CD45RC expression. CTL with tumor and allogeneic selectivity as well as SAg-induced CTL all expressed the LFA-1high phenotype, suggesting that upregulation of LFA-1 is a hallmark for in vivo activated CTL. RT-PCR analyses showed that all CD8+TCR alpha beta+ CTL lost expression of CD45R exon 4 mRNA, which is compatible with the view that effector/memory T cells are CD45RA-. In contrast, TCR gamma delta+ CTL retained the CD45RA phenotype but showed a down-regulation of CD45R exon 8 mRNA. Since isoforms of the CD45 tyrosine phosphatase have been reported to differentially affect T-cell activation, the unique CD45R splice pattern observed in TCR alpha beta+ and TCR gamma delta+ CTL implies that CD45R may influence the TCR signal transduction distinctly in various effector CTL subsets.

The protein tyrosine kinase p56lck regulates TCR expression and T cell selection.

The role of the protein tyrosine kinase (PTK), p56lck, in T cell development was evaluated by mating TCR transgenic mice with transgenic mice that expressed lckF505, a constitutively activated form of p56lck which is under the control of the lck proximal promoter element. The TCR transgenic mice expressed either a receptor specific for the male antigen presented by Db (H-Y TCR) or a receptor specific for pigeon cytochrome c peptide presented by I-Ek class II MHC molecules (AND TCR). The lckF505 transgene caused lower TCR expression in immature CD4+CD8+ thymocytes from normal and TCR transgenic mice. Consistent with the conclusion that activated p56lck causes lower TCR expression, the PTK inhibitor, herbimycin A, was able to restore TCR expression to normal levels in CD4+CD8+ thymocytes from TCR/lckF505 doubly transgenic mice. However, despite lower TCR expression, calcium mobilization was only moderately reduced in CD4+CD8+ thymocytes from H-Y TCR/lckF505 doubly transgenic mice. Furthermore, negative selection of CD4+CD8+ thymocytes expressing the H-Y TCR occurred efficiently in H-Y TCR/lckF505 doubly transgenic male mice despite lower TCR levels. By contrast, analysis of H-Y TCR/lckF505 and AND TCR/lckF505 doubly transgenic mice showed that positive selection in these mice was reduced by 4- to 5-fold by the lckF505 transgene. The smaller proportion of cells that were positively selected in doubly transgenic lckF505 mice expressed normal levels of TCR but higher levels of the appropriate CD4 or CD8 co-receptor molecule. These results indicate that the positive selection of thymocytes is regulated by the enzymatic activity of p56lck.

Different types of in vivo induced cytolytic T lymphocytes are all LFA-Ihigh and lack CD45 exon 4 (CD45RA) but show distinct CD45RC profiles.

Due to alternate mRNA splicing of exons 4, 5 and 6 (or A, B and C respectively), the CD45 cell surface glycoprotein is structurally heterogeneic in lymphoid cells of different lineage or stage of activation. Previous studies show that in vivo induced allo- and superantigen reactive rat cytolytic T lymphocytes (CTL) preferably belong to the CD45RClow subset, whereas tumour selective CTL express high amounts of CD45RC cell surface molecules. In this paper, reverse transcription polymerase chain reaction technique (RT-PCR) was utilized to evaluate CD45 isoform expression of rat lymphoid cells and in vivo activated rat CTL with distinct specificity and CD45RC profile. Cells from lymphoid organs expressed six CD45 mRNA isoforms, exon(45678), exon(5678), exon (578), exon(678), exon(78) and a novel extensively spliced exon(8) variant. In vivo activated TCR alpha beta + CD8+ cells sorted as CD45RClow expressed exon(78), exon(578) and exon(8), whereas TCR alpha beta + CD8+CD45RChigh cells expressed exon(78), exon(578), exon(5678) and full-length exon(45678). Triple-colour staining indicated high expression of LFA-1 in the cytotoxic CD45RCintermediate and CD45RClow cells, and low expression of LFA-1 in CD45RChigh non-cytotoxic cells from allo- and superantigen activated rats. In contrast, tumour activated TCR alpha beta +CD45RChigh cells were divided in LFA-1high and LFA-1low subsets, and sorting of these subsets revealed that tumour-selective cytotoxicity was confined to the LFA-1high effector cell subset. Furthermore, it was evident that the LFA-1high effector cell subset expressed high levels of exon(5678), exon(578) and exon(78) isoforms and, in contrast to the LFA-1low subpopulation, lacked expression of exon 4 containing full-length CD45 mRNA transcript.(ABSTRACT TRUNCATED AT 250 WORDS)

Locally superantigen-activated peritoneal cytolytic T lymphocytes belong to the CD8+ CD45RC- subset and lyse MHC class II+ tumor cells.

Bacterial encoded superantigens (SA) are capable of activating and targeting cytolytic human and mouse T lymphocytes (CTL) to lyse major histocompatibility complex class II positive (MHC class II+) target cells. In this study both in vitro and in vivo activated rat CTL were directed against MHC II+ tumor targets by bacterial encoded SA. Polyclonal in vitro activation of rat peripheral blood T lymphocytes generated CTL capable of killing MHC class II+ human BSM cells coated by staphylococcal enterotoxin (SE) -A, -E, -D, and TSST-1 but not by SEB or SEC1-3. Allo selective peritoneal CTL generated by intraperitoneal stimulation with allogeneic spleen cells were directed against BSM cells by SEA, -D, and -E but not by SEB, SEC1-3 or TSST-1. Based on the above observations, and in order to locally activate CTL, SEA was chosen for in vivo priming of rats by intraperitoneal inoculation of the toxin. SEA injection generated highly cytolytic CTL, and maximum cytolytic responses were seen at 50-250 micrograms SEA per animal with a peak in response 48-72 hours after injection of the toxin. The cytolytic activity of peritoneal SEA reactive effector cells was confined to the TCR alpha beta+ CD4- CD8+ CD45RC- cell population. MHC class II- colon carcinoma cells were insensitive to lysis by SEA reactive CTL but colon carcinoma cells induced to express MHC class II by interferon-gamma (IFN-gamma) treatment were efficiently lysed in the presence of SEA. Comparison of rat and human MHC II+ colon carcinomas revealed a peak in sensitivity to lysis at 10-100 ng SEA/ml for both tumor targets. These findings suggest that superantigens can be used in local immunotherapy of peritoneal tumors such as ovarian and colorectal carcinomatosis, with inducible or constitutive expression of MHC class II.

In vivo induced allo-reactive natural killer cells.

We have previously shown that rat allo-selective cells of the CD2+CD5- phenotype were generated in Brown Norway (BN) rats after immunization with allogeneic Wistar/Furth (WF) cells, whereas immunization with semi-allogeneic F1 (WF/BN) cells generated CD2+CD5+ effector T cells. We now report that the allo-selective CD2+CD5- lymphocytes lacked expression of intact CD3 complexes and expressed NKR-P1 molecules although lower as compared to classical NK cells, implicating that these lymphocytes constitute a subset of NK cells. The CD5+ T cells were not cytolytically active in BN rats immunized with WF cells indicating an intersubset regulation with mutually exclusive activation of either allo-selective T cells or allo-selective NK cells. Cold target inhibition showed that lysis of both allogeneic target cells and NK-sensitive target cells was mediated by the same NKR-P1 intermediate effector cells. These NK cells lysed WF but not allogeneic Fischer 344 or autologous BN target cells, indicating selective recognition of an allogeneic determinant. Semiallogeneic F1 (WF/BN) target cells were not lysed. Furthermore, target cells from F1 (WF/BN) x WF back-cross hybrids lacking expression of RT1n (self-MHC class I) were susceptible to lysis, whereas back-cross hybrids expressing RT1n were protected from lysis, indicating that self-MHC molecules conferred protection from lysis. These findings implicate the existence of NKR-P1intermediate and NKR-P1high NK cell subsets with different regulation and function in vivo.

In vivo induction of gamma/delta T cells with highly potent and selective anti-tumor cytotoxicity.

High frequencies of CD5+TcR alpha/beta- T cells were induced in the peritoneal cavity of rats immunized with syngeneic W439 lymphoma cells. These TcR alpha/beta- cells expressed TcR delta mRNA as analyzed by the polymerase chain reaction technique. The delta + (TcR gamma/delta +) T cells were of the CD2+, CD3+, CD4-, CD8+, CD45RB+ phenotype and showed stronger anti-tumor cytotoxicity compared to the TcR alpha/beta + T cells. The cytotoxic effects of both alpha/beta and gamma/delta T cells were selective for the W439 lymphoma cells and were not directed to other syngeneic tumors, natural killer targets and syngeneic or allogeneic normal cells. T cells, including both alpha/beta and gamma/delta cells, were induced when WF rats were immunized with allogeneic BN spleen cells. In this case the gamma/delta T cells showed allo-selective cytotoxicity, although weaker compared to the TcR alpha/beta + T cells. The gamma/delta T cells, induced by immunization with either W439 cells or BN spleen cells, were selective for the immunogen used and had no effect on irrelevant target cells, indicating that these effector cells were not activated by a shared gamma/delta T cell-related superantigen. Since highly potent tumor-selective gamma/delta cytotoxic T lymphocytes could be induced by syngeneic lymphoma cells, we suggest a role for gamma/delta T cells in the defense against certain types of tumors.

Functions of rat CD4+ T cell subsets defined by CD45RB: CD45RB- cells have a much stronger response to recall antigens, whereas polyclonally activated cells of both subsets are equally efficient producers of IFN in the presence of exogenous IL-2.

CD4+45RB- rat T cells were shown to respond strongly to recall antigens and produce IFN and TNF after polyclonal activation. Compared to CD4+45RB- cells, CD4+45RB+ cells showed a very weak response to recall antigens but produced higher amounts of IFN and TNF after polyclonal activation. Addition of rIL-2 reduced the difference between the subsets with respect to the level of IFN produced at 48 and 72 hr after activation, but did not influence the level of TNF production. The CD4+45RB- cells clearly showed a faster response to polyclonal activation than that of CD4+45RB+ cells detected as an earlier IFN production and CD25 expression. The earlier IFN production by the CD45RB- population could not only be explained by their faster production of IL-2, since the difference persisted when rIL-2 was added to both populations at the beginning of culture. We conclude that the CD4+45RB- rat T cell population resemble the CD4+45RA-0+ human T cell subset with respect to a good responsiveness to recall antigen and efficient production of IFN. However, the CD4+45RB+ rat T cell subset functionally differs from the CD4+45RA+0- human T cell subset. We suggest that the CD4+45RB+ subset comprises a major CD4+45RA+B+0- and a minor CD4+4+45A-B+0+ T cell subpopulation, the latter possibly mediating a response to recall antigen and the production of IFN.

Phenotypical and functional differentiation of CD4+ CD45RA+ human T cells following polyclonal activation.

Human CD4+ T cells differ in their expression of the leucocyte common antigen. Antibodies detecting certain forms (CD45RA and CD45RO) of this antigen have been used to identify and isolate subpopulations of the CD4+ T cells. These isolated subsets have been shown to have different abilities concerning lymphokine production and provision of help to B cells for Ig production. When these T-cell subsets were activated in vitro with polyclonal activators, the production. When these T-cell subsets were activated in vitro with polyclonal activators, the CD45RA+ cells lost this marker and gained the expression of CD45RO. This was true for all mitogens used in this report, i.e. accessory cell-dependent stimulation with SEA and accessory cell-independent activation with PMA or PHA. A correlation between proliferation and differentiation was observed, but this was probably not causative as stimulation with PMA in the absence of DNA synthesis resulted in the acquisition of CD45RO and loss of the CD45RA antigen. Moreover, cells proliferating vigorously for long periods of time expressed both markers at significant levels, which suggests that proliferation did not automatically result in complete loss of the CD45RA marker. The phenotypical differentiation was associated with a functional differentiation which induced the stimulated cells' ability to act as helper cells for Ig production and to produce gamma interferon (IFN-gamma). The results obtained in this study support the contention that the CD45RA+ cells are precursors of the CD45RO+ cells and that the two subsets represent different maturational stages of the same lineage.

Tumor-selective cytolysis is executed exclusively by CD45R+ CTL whereas allo-specific cytotoxicity can be executed also by CD45R- CTL.

Naive and memory CD4+ T helper cells can be distinguished on the basis of expression of the CD45R molecule. Whether this dichotomy applies also to CD8+ T cells has not yet been established. In the present investigation the cytolytic activity of peritoneal CD8+CD45R+ and CD8+CD45R- T cells from tumor- and allo-immunized rats has been studied. More than 90% of the CD8+ peripheral blood T lymphocytes expressed the CD45R molecule, whereas in the peritoneal cavity about 60% of the CD8+ T cells displayed the CD45R+ phenotype. Analysis of cytotoxicity of sorted peritoneal cells of W439 tumor-immunized donors demonstrated selective cytolytic activity of the CD5+CD4-CD8+CD45R+ subpopulation to W439 lymphoma target cells but no effect of CD5+CD4-CD8+CD45R- lymphocytes. None of these lymphocyte populations exhibited cytolytic activity to the NK-sensitive cell line YAC-1, whereas the CD5-CD45R+ population showed strong cytotoxicity to YAC-1 cells. In allo-immunized rats both CD5+CD4- CD8+CD45R+ and CD5+CD4-CD8+CD45R- peritoneal cells exhibited strong allo-specific cytolytic activity, but no activity to YAC-1 cells. Both CD5+CD4-CD8+CD45R+ and CD5+CD4-CD8+CD45R- cells from tumor-immunized rats proliferated in response to Con A and rIL-2. This is the first study demonstrating that tumor-selective cytolytic CD8+ T cells express the CD45R molecule and that allo-specific cytolytic CD8+ T cells are found in both the CD45R+ and CD45R- populations.

Gamma interferon induces the appearance of a CD4-, major histocompatibility complex class II+ macrophage subpopulation in the rat peritoneal cavity.

Peritoneal cells from gamma interferon (IFN-gamma)-treated rats were phenotypically characterized by flow cytometry. Intraperitoneal inoculation of 3 x 10(4) units of IFN-gamma induced, within 24 h, the appearance of a CD4-, major histocompatibility complex (MHC) class II+ macrophage subpopulation not present in rats treated with phosphate-buffered saline. IFN-gamma induced an increased expression of MHC class II I-A molecules on both CD4- and CD4+ macrophages. Both cell types adhered to plastic and expressed high levels of the macrophage membrane molecules CD11b and OX41. Histological examination of sorted CD4- and CD4+ macrophages confirmed the macrophage morphology of both populations with less granula in the former. We conclude that the appearance of CD4- macrophages in the peritoneal cavity after inoculation with IFN-gamma most probably reflects a selective recruitment of these cells from blood or surrounding tissues. The function of these cells is still unknown, although strong expression of MHC class II I-A indicates competence as antigen-presenting cells.

Rapid response to Con A by CD4+CD45R- rat memory lymphocytes as compared to CD4+CD45R+ lymphocytes.

Functionally distinct subpopulations within the CD4+ subset of T lymphocytes have been described in man, rat, and mouse. In the rat different functions have been assigned to CD45R+ and CD45R- T helper cells. The CD45R+ in contrast to the CD45R- T helper cells have been reported to produce IL-2 and to proliferate well in response both to Con A and in MLR. In the present investigation the kinetics of the response to Con A by the CD45R+ and CD45R- rat T helper subsets have been analyzed. We confirm a strong proliferative response to Con A by CD4+CD45R+ rat T lymphocytes and also that they are the best IL-2 producers. We further demonstrate that CD4+CD45R- cells also produce IL-2, although in order to appreciate this production quantitatively by assays of the culture supernatants it was necessary to block IL-2 absorption by IL-2 receptor (IL-2R) antibodies. This blockage was of importance also in comparisons of the two subsets, since they showed different kinetics of IL-2R appearance. It is demonstrated that the CD4+CD45R- cells respond more rapidly to Con A than the CD4+CD45R+ cells as reflected by phenotypic conversion, IL-2 production, and proliferation. The fast response of the CD4+CD45R- T subset shown in the present study of rat cells and analogous studies of human cells suggests that the memory compartment of T cells besides other characteristics also has the capacity for a more rapid response than naive lymphocytes.