CD3 Cell Surface Expression Research Articles

In vitro expansion of CMV-specific CD4+ T-cells by artificial antigen presentation for adoptive immunotherapy in the post- transplant setting

3049 Background: CMV continues to be a major cause of morbidity and mortality in marrow allograft recipients. The importance of CD4+ T-cells in maintaining immunity and therapeutic efficacy in adoptive cellular strategies has been appreciated. With a novel artificial antigen presenting cell (AAPC) system, we induced the expansion of CMV-specific CD4+ T-cells. Methods: AAPCs were generated by using a standardizable line of murine 3T3 cells sequentially transduced to express human ICAM-1, LFA-1, B7.1, HLA A*0201 and DRB1*1101 alleles. Antigen was provided either by peptide pulsing with the HLA-DRB1*1101-restricted, CMV- pp65-derived P92 peptide, or the influenza matrix peptide (IMP) as a control, or by transgene expression of pp65. PBMCs were isolated from a CMV+, HLA*A02, DRB1*11-positive healthy donor after informed consent, and enriched for T-cells via positive selection using sheep RBCs. Cocultures of T-cells with AAPCs were established at a ratio of 10:1. 20 IU/ml of IL-2 was added on d7 and every third day thereafter. On d12- 14 of co-culture, the T-cells were restimulated on a fresh monolayer of AAPCs with antigen. Cells were counted in triplicate via hemocytometer with trypan blue exclusion. Immunophenotyping of expanded T-cells was done via flow cytometric analysis for cell surface expression of CD3, CD8, CD4, and soluble Class II (HLA-DRB1*1101) tetramer specific for the P92 peptide of CMV-pp65. Results: Following 3 rounds of stimulation, antigen-specific fold expansion, defined by DR11,P92-tetramer-positive, CD4+ T-cells, was 313-fold when stimulated on AAPC.A2.DR11.PP65, compared to 5–35-fold using the other AAPC controls. Conclusion: We are able to expand clinically relevant numbers of CMV-specific CD4+ T-cells using a rapid, practicable, and broadly applicable approach. These preclinical studies show the feasibility of generating virus-specific T-cells of desired specificity and HLA restriction for adoptive immunotherapy of severe CMV infections. [Table: see text] No significant financial relationships to disclose.

Deficiency of decidual IL-10 in first trimester missed abortion: a lack of correlation with the decidual immune cell profile.

To determine if first trimester missed abortion decidua is characterized by an altered immune cell profile and/or a modified interleukin (IL)-10 and interferon (IFN)-gamma production pattern compared with decidua from elective termination. Flow cytometry and immunohistochemistry techniques were used to determine the decidual immune cell phenotypic profile and production pattern of IL-10 and IFN-gamma in cases of elective termination (n = 14) and missed abortion (n = 12). Both groups had a similar proportion of CD56+ CD16-, CD56+ CD16+, CD19+, CD3+, CD4+, CD8+, alphabeta T cells and gammadelta T cells. The majority of alphabeta and gammadelta positive T cells in both groups coexpressed the natural killer (NK) cell marker CD56, but lacked cell surface expression of CD3. Diminished decidual IL-10 staining was noted in 7/10 missed abortion cases compared with none of the elective termination cases (n = 12) (P = 0.007). A uniform decidual IFN-gamma staining pattern was observed in both groups. Decreased IL-10 production coupled with a sustained IFN-gamma presence noted in missed abortion compared with elective termination cases suggest that these cytokines may be important determinants in pregnancy outcome. In contrast, differences in the proportion of immune cells between both groups may not be a critical factor in early pregnancy loss. In normal pregnancy, decidual alphabeta and gammadelta positive T cells with reduced CD3 on their cell surface may be intrinsically restricted in T-cell receptor (TCR)-mediated activation.

Efficient class I major histocompatibility complex down-regulation by simian immunodeficiency virus Nef is associated with a strong selective advantage in infected rhesus macaques.

Substitution of Y223F disrupts the ability of simian immunodeficiency virus (SIV) Nef to down-modulate major histocompatibility complex (MHC) class I from the cell surface but has no effect on other Nef functions, such as down-regulation of CD4, CD28, and CD3 cell surface expression or stimulation of viral replication and enhancement of virion infectivity. Inoculation of three rhesus macaques with the SIVmac239 Y223F-Nef variant revealed that this point mutation consistently reverts and that Nef activity in MHC class I down-modulation is fully restored within 4 weeks after infection. Our results demonstrate a strong selective pressure for a tyrosine at amino acid position 223 in SIV Nef, and they constitute evidence that Nef-mediated MHC class I down-regulation provides a selective advantage for viral replication in vivo.

On the Dynamics of TCR:CD3 Complex Cell Surface Expression and Downmodulation

TCR downmodulation following ligation by MHC:peptide complexes is considered to be a pivotal event in T cell activation. Here, we analyzed the dynamics of TCR:CD3 cell surface expression on resting and antigen-activated T cells. We show that the TCR:CD3 complex is very stable and is rapidly internalized and recycled in resting T cells. Surprisingly, the internalization rate is not increased following TCR ligation by MHC:peptide complexes, despite significant TCR downmodulation, suggesting that constitutive internalization rather than ligation-induced downmodulation serves as the force that drives serial ligation. Furthermore, TCR downmodulation is mediated by the intracellular retention of ligated complexes and degradation by lysosomes and proteasomes. Thus, our data demonstrate that ligation induces TCR downmodulation by preventing recycling rather than inducing internalization.

Accumulation of Immature CD3 -CD4 +CD8 - Single-Positive Cells That Lack CD69 in Epithelial Cell Tumors of the Human Thymus

Human thymomas are epithelial tumors that are associated with a large number of nonneoplastic T cells of mainly immature phenotype, suggesting that epithelial cells of thymomas retain the function of thymic cortical epithelium. We report here that a large proportion (16-85%, mean ± SD 58 ± 22%, n = 17) of CD4 + single-positive T cells within the thymoma lack cell surface expression of CD3 and also of CD69, an early activation antigen that is expressed by positively selected thymocytes. Normal thymus had this CD4 +CD8 -CD3 - population at much lower levels (12 ± 7%, n = 11). It has been reported that in the human the CD4 +CD8 -CD3 - cells are the predominant population that is intermediate between the CD4 -CD8 - double-negative and CD4 +CD8 + double-positive stages. In contrast to CD4 + single-positive cells, most of the CD8 + single-positive cells in the thymoma as well as in the normal thymus expressed CD69 and a high level of CD3. A partial explanation of these observations is that the epithelial cells of thymoma were unable to positively select all the immature thymocytes generated in the thymoma. This relative inefficiency of positive selection could not be attributed solely to the paucity of MHC class II expression in the thymoma.

Cyclic AMP- and inositol phosphate-independent inhibition of Ca2+ influx by cholera toxin in CD3-stimulated Jurkat T cells. A study with a cholera toxin-resistant cell variant and the Ca2+ endoplasmic reticulum-ATPase inhibitor thapsigargin.

In this report, we describe a Jurkat cell variant, termed JCT8, the selection of which is based upon its resistance to cell-growth inhibition mediated by the holotoxin of Vibrio cholerae, cholera toxin (CT). JCT8 cells exhibit normal cAMP production in response to various cAMP inducers, including CT, together with conserved ADP ribosylation in vitro of G-protein Gs alpha by the A subunit of the toxin. However, after a 4-h pretreatment with CT, JCT8 cells have a conserved expression of cell-surface CD3 molecules. These effects are in contrast to those elicited by the toxin in long term PGE2-desensitized Jurkat cells, which remain as sensitive as the wild type to the inhibitory action of CT on cell growth and CD3 cell-surface expression, despite poor responsiveness to CT with regard to cAMP production. In JCT8 cells, Ca2+ mobilization induced via the CD3/TCR is maintained after CT treatment contrasting with its complete suppression in the wild-type and in the PGE2-desensitized cells. However, as in the other cell types, CT still suppresses Ca2+ influx in JCT8 cells. Increase in inositol phosphates by CD3 stimulation of JCT8 cells, including of inositol 1,4,5-triphosphate (I(1,4,5)P3), is only partially antagonized by CT. This suggests either that in JCT8 cells there is a different susceptibility of Ca2+ mobilization and influx to partial inhibition by CT of CD3-triggered phospholipase C (PLC)-induced phosphoinositide hydrolysis or that an additional and PLC-independent suppressive effect of the toxin on Ca2+ influx may exist. To investigate this particular point further, we use Thapsigargin, a Ca(2+)-endoplasmic reticulum ATPase inhibitor that can mobilize in human T lymphocytes I(1,4,5)P3-dependent intracellular Ca2+ pools by a PLC-independent pathway. We demonstrate that the Ca2+ influx triggered in the wild-type Jurkat cells or in JCT8 cells by Thapsigargin is antagonized by CT. The present data are therefore consistent with the idea that CT specifically impairs in the Jurkat T cell model the entry of Ca2+ from extracellular spaces by a mechanism independent not only from cAMP but also in part from inhibition by the toxin of phosphoinositide hydrolysis.

Cell surface comodulation of CD4 and T cell receptor by anti-CD4 monoclonal antibody.

In our study we have used anti-CD4 mAb to investigate the cell surface association between CD4 and the Ag-specific TCR complex on mature peripheral T cells. Anti-CD4 mAb was administered in vivo and in vitro and its effects on CD4 and CD3 cell surface expression were determined. In vivo, anti-CD4 mAb reduced cell surface expression of its ligand, CD4, and secondarily also reduced cell surface expression of CD3/TCR on CD4+ splenic T cells. In vitro, multivalent cross-linking of CD4 by anti-CD4 mAb and either FcR+ cells or anti-Ig mAb also resulted in decreased surface expression of CD4 and specific comodulation of CD3/TCR. The secondary reduction in cell surface CD3/TCR expression induced by CD4 cross-linking could be pharmacologically disrupted by high doses of PMA, indicating that the comodulation of CD3 with CD4 was dependent upon intracellular mediators, possibly including protein kinase C. These results demonstrate that, in the presence of anti-CD4 mAb, CD4 is functionally associated with the CD3/TCR complex, and that this association is dependent upon the activity of intracellular mediators. Such intracellular mediators might induce the coordinate down-modulation of physically unassociated CD4 and CD3/TCR molecules, or, alternatively, might promote a physical interaction between CD4 and CD3/TCR molecules.

CD5 antibodies increase intracellular ionized calcium concentration in T cells.

The binding of a variety of monoclonal antibodies to the CD5 (T, gp67) pan T cell differentiation antigen has been shown to potentiate T cell proliferation. In this paper we show that CD5 monoclonal antibodies cause increased intracellular free calcium concentration ([Ca2+]i) in T cells. An increase in [Ca2+]i occurred within 1 min in indo-1-loaded PBMC after the addition of CD5 monoclonal antibodies and cross-linking with a second step anti-mouse kappa light chain antibody. Cross-linking of CD5 was effective when done directly on the cell surface or by the administration of preformed soluble complexes that contained CD5 antibodies. Calcium mobilization induced by suboptimal concentrations of CD3 antibodies was specifically augmented and sustained by CD5 antibodies, although the enhancement was modest in magnitude. When cell surface phenotype was correlated with calcium mobilization, it was found that the CD5 response was restricted to CD5+/CD3+ cells, and that approximately 90% of CD5+ cells had responded. CD5-induced calcium mobilization was found to differ from CD3 stimulation in that EGTA entirely ablated the CD5 response, whereas the CD3 response was resistant to EGTA, indicating that the CD5-induced increased [Ca2+]i is derived primarily or entirely from extracellular calcium. CD5-stimulated calcium mobilization also differed from CD3 in that the CD5 response was inhibited by pretreatment with phorbol myristate acetate, whereas the CD3 response was not, suggesting that depletion of protein kinase C causes an uncoupling of signal transduction between CD5 and calcium channels. Finally, experiments were done with T cells after antigenic modulation of the CD3 or CD5 molecules. Unexpectedly, both the CD5 and the CD3 responses were ablated on CD3-modulated cells, whereas only the CD5 response was ablated on CD5-modulated cells. In addition, several Cd5+/CD3- T cell leukemia lines also failed to respond to CD5 stimulation, providing further evidence which indicates that the CD5 response depends on the cell surface expression of CD3 or a CD3-associated structure. These findings suggest that one mechanism for CD5-induced augmentation of mitogen-stimulated T cell proliferation involves increased [Ca2+]i which is distinct from but interdependent with that induced by stimulation of the CD3 molecule.

Distinct patterns of transmembrane calcium flux and intracellular calcium mobilization after differentiation antigen cluster 2 (E rosette receptor) or 3 (T3) stimulation of human lymphocytes.

We evaluated CD2 (E rosette) and CD3 (T3)-triggered activation of resting lymphocytes by measuring the intracellular free calcium concentration ([Ca2+]i) of individual cells. The [Ca2+]i of indo-1-loaded cells was measured by flow cytometry and responses were correlated with cell surface phenotype. Stimulation with anti-CD3 antibody caused an increase in [Ca2+]i in greater than 90% of CD3+ cells within 1 min, and furthermore, the response was restricted to cells bearing the CD3 marker. In contrast, stimulation of cells with anti-CD2 antibodies produced a biphasic response pattern with an early component in CD3- cells and a late component in CD3+ cells. Thus, the CD2 response does not require cell surface expression of CD3. In addition, stimulation of a single CD2 epitope was sufficient for activation of CD3- cells, whereas stimulation of two CD2 epitopes was required for activation of CD3+ cells. Both the CD2 and CD3 responses were diminished in magnitude and duration by EGTA. However, approximately 50% of T cells still had a brief response in the presence of EGTA, indicating that the increased [Ca2+]i results in part from intracellular calcium mobilization, and furthermore demonstrates that extracellular calcium is required for a full and sustained response. Our results support the concept that CD2 represents the trigger for a distinct pathway of activation both for T cells that express the CD3 molecular complex and for large granular lymphocytes that do not.