Differential interleukin secretion by in vitro activated human CD45RA and CD45RO CD4+ T cell subsets.

The CD45RA and CD45RO isoforms have been reported to define complementary subsets among CD4+ T cells: CD45RA CD4+ T cells are considered "virgin T cells" and CD45RO "primed T cells." We investigated the secretion of lymphokines by human CD4+ CD45RO and CD4+ CD45RA T helper cells after mitogen stimulation. CD45RA and CD45RO CD4+ T cells were isolated by negative immunoselection using magnetic beads. CD45RO cells, but not CD45RA cells, proliferate well in response to pokeweed mitogen (PWM) or insoluble anti-CD3. Both subpopulations produced interleukin (IL)-2, IL-6, and interferon (IFN)-gamma when stimulated with PWM for 1-4 days. Only Day 1 supernatants from CD45RO cells contained moderate amounts of IL-4. After 14 days of continuous culture and stimulation with PWM, the CD45RA subset had lost the expression of CD45RA and gained that of CD45RO. When long-term cultured CD45RA or CD45RO cells were treated with insoluble anti-CD3, they incorporated [3H]thymidine at similar levels, but only CD45RO cells secreted IL-4 and significantly increased their secretion of IFN-gamma. These data indicate that despite phenotype conversion, the two subpopulations maintain functional differences in the secretion of lymphokines, thus suggesting that circulating CD45RA and CD45RO cells may represent different lines of differentiation.     

CD4+CD45RA+ and CD4+CD45RA- T cell subsets in man maintain distinct function and CD45RA expression persists on a subpopulation of CD45RA+ cells after activation with Con A.

We have previously shown that Con A-induced suppressor T cells belong to the CD45RA+ subset. After unseparated T cells are activated with Con A, CD45RA expression increases to a maximum (Day 2), and then decreases significantly, but does not disappear entirely (Day 9), while CD29 expression increases steadily. In the present study, we examined the fate of these cell surface molecules on isolated CD4+CD45RA+ and CD4+CD45RA- cells following activation with Con A, and their relationship to the regulatory functions of these subsets. After activation of CD4+CD45RA+ cells with Con A, CD45RO and CD29 antigen expression rapidly increases (greater than 90%). While CD45RA expression is downregulated, approximately 40% of the cells continue to express low-density CD45RA in a stable fashion through Day 21. Despite these phenotypic changes, cells originally CD45RA+ continue to suppress IgG synthesis and provide only minimal B cell help. Furthermore, when cells originally CD45RA+ were sorted on the basis of continued presence, or loss of CD45RA antigen 14 days after activation, both populations demonstrated potent suppression and minimal help. In contrast, after activation with Con A, CD4+CD45A- cells maintain stable phenotype and provide significant help and minimal suppression. Immunoprecipitation of the CD45RA antigen from Day 14 activated CD4+CD45RA+ cells confirms the continued presence of the 205-kDa isoform, but reveals a significant decrease in the 220-kDa isoform. These results suggest that after activation with Con A, cells originally CD45RA+ remain functionally distinct from cells originally CD45RA-, and that CD45RA antigen persists on a subpopulation of CD45RA+ cells after activation with Con A.     

Either of the CD45RB and CD45RO isoforms are effective in restoring T cell,but not B cell,development and function in CD45-null mice

The protein tyrosine phosphatase CD45 is expressed as a series of isoforms whose tissue and differentiation stage specificity is broadly conserved in evolution. CD45 has been shown to be an important regulator of a variety of functions in many different hemopoietic lineages. We have chosen an in vivo genetic complementation strategy to investigate the differential functions between isoforms. In this study, we report the characterization of transgenic mice which express the isoforms CD45RO or CD45RB as their only CD45 molecules, at a variety of expression levels and in the majority of hemopoietic lineages. Both CD45RO and CD45RB isoforms reconstitute thymocyte development in a CD45-null mouse background when expressed above a threshold level. The resulting mature T cells populate the peripheral lymphoid organs where they are found at normal frequency. Both CD45RO and CD45RB isoforms also permit T cell function in the periphery, although the threshold for normal function here appears to be set higher than in the thymus. In contrast, neither isoform is capable of fully restoring peripheral B cell maturation, even at levels approaching those in heterozygous CD45(+/-) mice in which maturation is normal. In vitro activation of B cells by Ag-receptor stimulation is only minimally complemented by these CD45RO and CD45RB transgenes. Our results suggest that CD45 isoforms play unique roles which differ between the T and B lineages.     

Human CD8+ T lymphocytes can be divided into CD45RA+ and CD45RO+ cells with different requirements for activation and differentiation.

The functional distinction between CD45RA+ and CD45RO+ cells within the human CD4+ T cell subset is well established. This study was undertaken to investigate whether a similar division can be made within the CD8+ T cell population. A quantitative comparison was made of the requirements for activation and differentiation of CD8+CD45RA+ and CD8+CD45RO+ cells. Stimulation of T lymphocytes with anti-CD3 mAb immobilized at high-density induced strong proliferation and CTL activity in both CD45RA+ and CD45RO+ cells. Suboptimal TCR/CD3 triggering, in contrast, induced substantially higher levels of proliferation and CTL activity in CD8+CD45RO+ cells compared with their CD45RA+ counterparts. Lymphokine secretion (i.e., Il-2 and TNF-alpha) was under any condition more readily induced in CD8+CD45RO+ cells. Markedly, proliferation of both CD8+CD45RA+ and CD8+CD45RO+ T cells initiated by anti-CD3 mAb immobilized at high densities was not inhibited by addition of anti-CD25 mAb, in contrast to proliferation induced by suboptimal anti-CD3 mAb concentrations. These findings show that a functional division between CD45RA+ and CD45RO+ T cells with distinct requirements for activation and differentiation may also be made in the CD8+ subset.     

Phenotypic comparison of the three populations of human lymphocytes defined by CD45RO and CD45RA expression.

Published reports indicate that CD45RO-CD45RAbright T cells are native T cells, CD45RObrightCD45RA- T cells are memory T cells, and that concomitant loss of CD45RA expression and gain of CD45RO expression occurs during transition from naive to memory status. Thus, following in vitro activation of CD45RO- CD45RAbright T cells, a subset of transitional CD45ROdimCD45RAdim T cells is observed before conversion to a CD45RObrightCD45RA- phenotype is completed. Interestingly, all three of these phenotypic subsets are represented in the circulating human lymphocyte pool. We thus used dual-color flow cytometry to phenotypically characterize CD45RObrightCD45RA-, CD45ROdimCD45RAdim, and CD45RO- CD45RAbright lymphocytes. Both the CD45RObrightCD45RA- and CD45ROdimCD45RAdim subsets consisted almost entirely of T cells, whereas the CD45RO-CD45RAbright subset contained T cells plus essentially all of the B and natural killer cells. Additional studies used three-color flow cytometry to assess activation markers on T cells within the three subsets defined by CD45RO/CD45RA expression. CD25 expression increased with conversion from naive to memory status (5% of CD45RO-CD45RAbright, 24% of CD45ROdimCD45RAdim, and 42% of CD45RObrightCD45RA- T cells), whereas CD38 expression decreased during conversion (76, 53, and 27%, respectively). We also assessed the fluorescent intensities of CD11a, CD2, and CD44, shown by others to be increased on memory, compared to naive T cells. Visual inspection of fluorescence cytograms confirmed these findings, and further showed that transitional T cells express these markers at levels indistinguishable from those for naive T cells. These findings suggest that acquisition of CD25 and loss of CD38 occur relatively early in the naive-to-memory transition process, being evident in the transitional cell subset. In contrast, increased expression of CD11a, CD2, and CD44 appear to represent late events, occurring after loss of CD45RA and gain of CD45RO has been completed.     

Immunophenotypic Characterization of CD45RO+ and CD45RA+ T Cell Subsets in Peripheral Blood of Peripheral T Cell Lymphoma Patients

To study the distribution profile of CD45RO⁺ and CD45RA⁺ T cells in the peripheral blood of peripheral T cell lymphoma (PTCL) patients and its clinical significance. 27 patients with PTCL were enrolled in this study, together with 30 healthy individuals as the control group. Flow cytometry analysis was employed to examinate the differences in the distribution of CD45RO⁺ and CD45RA⁺ T cells in peripheral blood between two groups. In PTCL patient’s lymphnode tissues, the T cell population displayed diverse antigenic expression, with CD4⁺ T cells as the major subset. No B cell-related antigen was expressed. The percentage of CD4⁺/CD8⁺ and CD4⁺CD45RO⁺ T cells in patients’ peripheral blood were significantly lower than that in the control samples, while the percentage of CD4⁺CD45RA⁺, CD8⁺CD45RA⁺, and CD8⁺CD45RO⁺ T cells in patients’ peripheral blood were significantly higher than that in the control samples. The percentage of CD4⁺/CD8⁺, CD4⁺CD45RO⁺ cells in stage I/II PTCL patients’ peripheral blood were significantly higher than that in the samples from patients with stage III/IV PTCL. The percentage of CD4⁺CD45RA⁺, CD8⁺CD45RA⁺, and CD8⁺CD45RO⁺ T cells were notably lower than that in the samples from III/IV period PTCL patients. Both CD45RO⁺ and CD45RA⁺ T cells play important roles in the process of PTCL. The immunophenotypic profile from this study will help to develop the differential diagnosis and treatment of PTCL patients in the future, and improve the accuracy rate of diagnosis and to ameliorate the prognosis.     

Specific CD45 isoforms differentially regulate T cell receptor signaling.

Multiple isoforms of T cell CD45 tyrosine phosphatase are expressed as a result of alternative RNA splicing among extracellular exons. To discern the presence and identity of distinct functions among CD45 isoforms, we compared thymic T cell activation responses by elevating expression of two CD45 isoforms normally found on quiescent T cells. We report that CD45RABC significantly increased CD4+ thymic T cell proliferation in both a mixed lymphocyte reaction and following anti-T cell receptor (TCR) antibody stimulation. Additionally, CD45RABC enhanced Ca2+ mobilization and phosphotyrosine accumulation, and suppressed the inhibitory effect of anti-CD4 antibodies. By contrast, CD45R0 did not enhance TCR signaling or phosphotyrosine levels in CD4+ thymic T cells and required a TCR co-stimulus to augment cellular proliferation. These studies provide genetic evidence that alternative CD45 isoforms are functionally distinct and disclose a unique mechanism by which T cell immunologic responsiveness can be modified.     

alpha2,6-Sialylation promotes binding of placental protein 14 via its Ca2+-dependent lectin activity: insights into differential effects on CD45RO and CD45RA T cells

Placental protein 14 (PP14; glycodelin) is a pregnancy-associated immunoregulatory protein that is known to inhibit T cells via T-cell receptor desensitization. The recent demonstration of PP14 as lectin has provided insight into how it may mediate its CD45 glycoprotein-dependent T-cell inhibition. In this study, we have investigated PP14's lectin-binding properties in detail. Significantly, PP14 reacts with N-acetyllactosamine (LacNAc) as was also found for members of the galectin family, such as the potent immunoregulatory protein, galectin-1. However, in contrast to galectin-1, PP14's binding is significantly enhanced by alpha2,6-sialylation and also by the presence of cations. This was demonstrated by preferential binding to fetuin as compared with its desialylated variant asialofetuin (ASF) and by using free alpha2,6- versus alpha2,3-sialylated forms of LacNAc in competitive inhibition and direct solid-phase binding assays. Interestingly, from immunological point of view, PP14 also binds differentially to CD45 isoforms known to differ in their degree of sialylation. PP14 preferentially inhibits CD45RA+, as compared with CD45RO+ T cells, and preferentially co-capped this variant CD45 on the T-cell surface. Finally, we demonstrate that PP14 promotes CD45 dimerization and clustering, a phenomenon that may regulate CD45 activity.     

CD45 isoform expression on human neonatal T cells: expression and turnover of CD45 isoforms on neonatal versus adult T cells after activation.

Neonatal T cells are phenotypically similar to "naive" T cells from adult donors in the CD45 isoform expression. Despite the phenotypic similarity, large differences were found between neonatal and adult T cells when T cells were activated. After activation with PHA, adult CD45RA+ T cells began to express CD45RO and no loss of CD45RA expression had yet occurred at Day 3 post-stimulation. Three days after activation, CD45RA+ neonatal T cells also coexpressed CD45RO; however, in contrast to adult T cells, a marked loss of CD45RA was observed. We analyzed the rapid loss of CD45RA found in neonatal T cells. The de novo synthesis of CD45 isoforms in neonatal T cells was essentially the same as that in the adult T cells. Turnover of the CD45RA was very rapid in both resting adult and neonatal T cells. After activation with PHA, the turnover of CD45RA on adult T cells was decreased significantly, while the turnover of CD45RA on neonatal T cells was not changed after activation. Therefore, the regulation of CD45 isoform expression not only involves switches in alternative splicing, but also involves different regulation of turnover of these isoforms from the cell membrane.     

Human autoreactive CD4+ T cells from naive CD45RA+ and memory CD45RO+ subsets differ with respect to epitope specificity and functional antigen avidity

T cells with specificity for self-Ags are normally present in the peripheral blood, and, upon activation, may target tissue Ags and become involved in the pathogenesis of autoimmune processes. In multiple sclerosis, a demyelinating disease of the CNS, it is postulated that inflammatory damage is initiated by CD4+ T cells reactive to myelin Ags. To investigate the potential naive vs memory origin of circulating myelin-reactive cells, we have generated myelin basic protein (MBP)- and tetanus toxoid-specific T cell clones from CD45RA+/RO- and CD45RO+/RA- CD4+ T cell subsets from the peripheral blood of multiple sclerosis patients and controls. Our results show that 1) the response to MBP, different from that to TT, predominantly emerges from the CD45RA+ subset; 2) the reactivity to immunodominant MBP epitopes mostly resides in the CD45RA+ subset; 3) in each individual, the recognition of single MBP epitopes is skewed to either subset, with no overlap in the Ag fine specificity; and 4) in spite of a lower expression of costimulatory and adhesion molecules, CD45RA+ subset-derived clones recognize epitopes with higher functional Ag avidity. These findings point to a central role of the naive CD45RA+ T cell subset as the source for immunodominant, potentially pathogenic effector CD4+ T cell responses in humans.     

T8 cell regulation of human B cell responsiveness: regulatory influences of CD45RA+ and CD45RA- T8 cell subsets

The immunoregulatory functions of human T8 cell subpopulations defined by mAb to the CD45RA molecule (2H4) were examined. Both CD45RA+ and CD45RA- T8 cells that had been treated with mitomycin C provided help for the production of immunoglobulins by B cells in cultures stimulated with immobilized mAb to CD3 (64.1). In contrast, both CD45RA+ and CD45RA- T8 cells that had not been treated with mitomycin C suppressed B cell responses in anti-CD3-stimulated cultures, although CD45RA+ T8 cells were more effective in this regard. Interleukin 2 (IL2) enhanced suppression by anti-CD3-activated CD45RA- T8 cells, whereas suppression by CD45RA+ T8 cells was almost maximal and not as much increased by IL2. The differentiation into suppressor-effector cells in this system appeared to involve the production of IL2, but not the production of interferon (INF)-gamma. Thus, CD45RA+ T8 cells produced higher amounts of IL2 but lower amounts of IFN-gamma than CD45RA- T8 cells in anti-CD3-stimulated cultures. Moreover, addition of mAb to the p55 component of IL2 receptor (anti-Tac) inhibited the generation of suppressor activity from CD45RA+ and CD45RA- T8 cells. The pattern and magnitude of suppression of B cell responses by CD45RA+ and CD45RA- T4 cells were similar to that by CD45RA+ and CD45RA- T8 cells in this system. Finally, preactivated CD45RA+ T8 cells that had lost CD45RA expression suppressed the B cell responses as effectively as fresh CD45RA+ T8 cells. The results indicate that both CD45RA+ and CD45RA- T8 cells can help or suppress B cell responses. More importantly, the data suggest that the suppressor-effector function of human T cells may rather be related with the stages of the post-thymic differentiation as evidenced by the expression of the CD45RA molecule than represent the fully differentiated T cell subsets, such as T4 and T8 cells. In addition, the CD45RA molecule appeared not to be involved in the suppressor-effector function, but to determine the stage of post-thymic differentiation.     

T cell proliferative response to interleukin 2: different frequency of responders among CD45R0 and CD45RA subsets

A proportion of human blood T cells is known to proliferate in cultures containing 10 or more units/ml of interleukin (IL)2, in the absence of exogenously added antigen. A possible explanation for this phenomenon is that the IL2 serves to maintain the proliferation of memory T cells which have recently been activated by antigen in vivo. To test this possibility we therefore separated T cells according to their CD45 (common leukocyte antigen) phenotype into the CD45RO and CD45RA subsets which are associated with memory and naive status, respectively. The frequency of responder cells in limiting dilution analysis of adult MNC was 1:1625 CD45RO cells, 1:2405 for CD45RA cells, and 1:1642 for unseparated cells. CD45RA cells from newborns had a 1:1850 frequency of responders. The IL2 responder cells were predominantly CD3+ TcR alpha beta +, with a minor component of TcR delta cells. There were no differences in the frequency of T cells using V beta 5 and V beta 8 between responding and nonresponding cells. The data argue against a requirement for prior sensitization for a proliferative response to IL2 and give no evidence that the subset of T cells which responds to IL2 differs in phenotype from nonresponsive cells.     

Vpr cytopathicity independent of G2/M cell cycle arrest in human immunodeficiency virus type 1-infected CD4+ T cells

The mechanism of CD4(+) T-cell depletion in human immunodeficiency virus type 1 (HIV-1)-infected individuals remains unknown, although mounting evidence suggests that direct viral cytopathicity contributes to this loss. The HIV-1 Vpr accessory protein causes cell death and arrests cells in the G(2)/M phase; however, the molecular mechanism underlying these properties is not clear. Mutation of hydrophobic residues on the surface of its third alpha-helix disrupted Vpr toxicity, G(2)/M arrest induction, nuclear localization, and self-association, implicating this region in multiple Vpr functions. Cytopathicity by virion-delivered mutant Vpr protein correlated with G(2)/M arrest induction but not nuclear localization or self-association. However, infection with whole virus encoding these Vpr mutants did not abrogate HIV-1-induced cell killing. Rather, mutant Vpr proteins that are impaired for G(2)/M block still prevented infected cell proliferation, and this property correlated with the death of infected cells. Chemical agents that inhibit infected cells from entering G(2)/M also did not reduce HIV-1 cytopathicity. Combined, these data implicate Vpr in HIV-1 killing through a mechanism involving inhibiting cell division but not necessarily in G(2)/M. Thus, the hydrophobic region of the third alpha-helix of Vpr is crucial for mediating G(2)/M arrest, nuclear localization, and self-association but dispensable for HIV-1 cytopathicity due to residual cell proliferation blockade mediated by a separate region of the protein.     

In vivo ultraviolet-exposed human epidermal cells activate T suppressor cell pathways that involve CD4+CD45RA+ suppressor-inducer T cells

In vivo UV exposure of human epidermis abrogates the function of CD1+DR+ Langerhans cells and induces the appearance of CD1-DR+ Ag-presenting macrophages. Epidermal cells from UV-exposed skin, in contrast to epidermal cells from normal skin, potently activate autologous CD4+ T cells, and, in particular, the CD45RA+ (2H4+) (suppressor-inducer) subset. We therefore determined whether UV-exposure in humans leads to a T cell response in which suppression dominates. Autologous blood T cells were incubated with epidermal cell suspensions from in vivo UV-irradiated skin. After activation, repurified T cells were transferred in graded numbers to autologous mononuclear cells (MNC) stimulated with PWM and the resultant IgG production analyzed by ELISA. Relative to T cells activated by unirradiated control epidermal cells, T cells activated by UV-exposed epidermal cells demonstrated enhanced capacity to suppress IgG production (n = 6; p less than or equal to 0.03). Within the T cell population, CD8+ cells stimulated by UV-exposed epidermal cells could be directly activated to suppress PWM-stimulated MNC Ig production if IL-2 was provided in the reaction mixture. The suppressive activity was also transferable with purified CD4+ T cells stimulated by UV-exposed epidermal cells (n = 10; p less than or equal to 0.01), and was radiosensitive. Suppression was decreased when PWM-stimulated MNC were depleted of CD8+ T cells before mixing with CD4+ T cells activated by UV-exposed epidermal cells, suggesting indirect induction of CD8+ Ts cells contained within the responding MNC populations. Indeed, physical depletion of CD45RA+ cells resulted in total abrogation of the suppressor function contained in the CD4+ T cells. Activation of suppressor function was critically dependent on DR+ APC contained in UV-exposed epidermis. The data suggest that UV-exposure modulates cutaneous APC activity in humans, as in mice, such that the dominant immune response is tilted toward suppression. These mechanisms in normal individuals may function to dampen responses to UV-induced endogenous Ag that are pathogenic in autoimmune disorders. However, these mechanisms might also facilitate the growth of UV-induced skin cancers.     

Beta-catenin regulation during the cell cycle: implications in G2/M and apoptosis

Beta-catenin is a multifunctional protein involved in cell-cell adhesion and Wnt signal transduction. Beta-catenin signaling has been proposed to act as inducer of cell proliferation in different tumors. However, in some developmental contexts and cell systems beta-catenin also acts as a positive modulator of apoptosis. To get additional insights into the role of beta-catenin in the regulation of the cell cycle and apoptosis, we have analyzed the levels and subcellular localization of endogenous beta-catenin and its relation with adenomatous polyposis coli (APC) during the cell cycle in S-phase-synchronized epithelial cells. Beta-catenin levels increase in S phase, reaching maximum accumulation at late G2/M and then abruptly decreasing as the cells enter into a new G1 phase. In parallel, an increased cytoplasmic and nuclear localization of beta-catenin and APC is observed during S and G2 phases. In addition, strong colocalization of APC with centrosomes, but not beta-catenin, is detected in M phase. Interestingly, overexpression of a stable form of beta-catenin, or inhibition of endogenous beta-catenin degradation, in epidermal keratinocyte cells induces a G2 cell cycle arrest and leads to apoptosis. These results support a role for beta-catenin in the control of cell cycle and apoptosis at G2/M in normal and transformed epidermal keratinocytes.     

Viral infections and cell cycle G2／M regulation

Progression of cells from G2 phase of the cell cycle to mitosis is a tightly regulated cellular process that requires activation of the Cdc2 kinase, which determines onset of mitosis in all eukaryotic cells. In both human and fission yeast(Schizosaccharomyces pombe) cells, the activity of Cdc2 is regulated in part by the phosphorylation status of tyrosine 15 (Tyrl5) on Cdc2, which is phosphorylated by Weel kinase during late G2 and is rapidly dephosphorylated by the Cdc25 tyrosine phosphatase to trigger entry into mitosis. These Cdc2 regulators are the downstream targets of two wellcharacterized G2/M checkpoint pathways which prevent cells from entering mitosis when cellular DNA is damaged or when DNA replication is inhibited. Increasing evidence suggests that Cdc2 is also commonly targeted by viral proteins,which modulate host cell cycle machinery to benefit viral survival or replication. In this review, we describe the effect of viral protein R (Vpr) encoded by human immunodeficiency virus type 1 (HIV-Ⅰ) on cell cycle G2/M regulation. Based on our current knowledge about this viral effect, we hypothesize that Vpr induces cell cycle G2 arrest through a mechanism that is to some extent different from the classic G2/M checkpoints. One the unique features distinguishing Vpr-induced G2 arrest from the classic checkpoints is the role of phosphatase 2A (PP2A) in Vpr-induced G2 arrest.Interestingly, PP2A is targeted by a number of other viral proteins including SV40 small T antigen, polyomavirus T antigen, HTLV Tax and adenovirus E4orf4. Thus an in-depth understanding of the molecular mechanisms underlying Vpr-induced G2 arrest will provide additional insights into the basic biology of cell cycle G2/M regulation and into the biological significance of this effect during host-pathogen interactions.     

Gadd45-induced cell cycle G2/M arrest for improved transient gene expression in Chinese hamster ovary cells

Gadd45 is a p53-regulated protein and is involved in cell cycle arrest in the G2/M phase. In an effort to improve transient gene expression (TGE) in Chinese hamster ovary (CHO) cells, the effect of Gadd45-induced cell cycle arrest on TGE in CHO cells was investigated using the two different expression vectors encoding Fcfusion protein and recombinant antibody. To regulate the expression of Gadd45 in CHO cells, the CHO-TREx-gadd45 cell line was established using the T-REx system controlled by doxycycline. During the cultures for TGE, Gadd45 overexpression severely inhibited cell growth, but significantly enhanced TGE. Compared with the culture without Gadd45 overexpression, the TGE of Fc fusion protein and humanized antibody were increased by 111 and 93%, respectively. The enhanced TGE, despite the cell growth arrest induced by Gadd45 overexpression, was due to the significantly increased specific productivity, resulting from enhanced transfection efficiency, increased cell size, and active DNA demethylation. Taken together, the data obtained here demonstrate that Gadd45-induced cell cycle arrest in G2/M phase can significantly enhance TGE in CHO cells.     

Bromelain treatment of human T cells removes CD44, CD45RA, E2/MIC2, CD6, CD7, CD8, and Leu 8/LAM1 surface molecules and markedly enhances CD2-mediated T cell activation.

Treatment of T cells with the cysteine protease bromelain has been widely used to enhance the binding of human T cells to human E (autologous E rosettes) and has been shown to remove surface T cell CD44 molecules. Ligand binding to CD44 has been shown to markedly augment T cell activation. To study the activation potential of bromelain-treated CD44 T cells, we have compared the proliferation of sham- and bromelain-treated normal human PBMC to mitogenic CD2 mAb. We found that bromelain not only removed T cell CD44, but also removed the CD45RA isoform of CD45 as well as E2/MIC2, CD6, CD7, CD8, and Leu 8/LAM1 molecules. T cell proliferation in response to CD2 mAb was increased 325% in bromelain-treated PBMC compared to sham-treated PBMC (p < 0.005). Reciprocal treatment experiments using purified T cells and monocytes demonstrated that the enhancement of T cell CD2 activation by bromelain occurred only when T cells were treated with bromelain and was accompanied by increased adhesion of T cells to monocytes. These data demonstrate that expression of portions of the extracellular domains of the CD44, CD45RA, E2/MIC2, CD6, CD7, CD8, and Leu 8/LAM1 surface molecules are not required for CD2 activation of human T cells. Rather, the removal of these surface molecules by bromelain is associated with enhanced T cell-monocyte aggregation and enhanced CD2-mediated T cell activation. Taken together with data that CD44, E2/MIC2, CD6, and CD7 mAb inhibit CD2/lymphocyte function-associated Ag-3-mediated cellular interactions and also augment CD2-mediated triggering of T cells, these data suggest that members of the bromelain-sensitive group of surface molecules may comprise a set of CD2-associated adhesion ligands that acts in concert to modulate human T cell activation.