Different effects of IL-2 addition or antibody crosslinking on T-cell subset stimulation by CD3 antibodies

The effect of exogenous recombinant interleukin-2 (IL-2) or of antibody crosslinking on the activation of human T-cell subsets by IgG2a (OKT3/BMA030), IgG1 (Leu4 and UCHT1), or IgG2b (BMA031) anti-T3 antibodies (CD3) was investigated. In so-called nonresponder cultures as well as in monocyte-depleted cell cultures addition of IL-2 increased the CD3-induced activation and proliferation of T4 and T8 cell subsets. Relatively more T8 than T4 cells were stimulated by antibody binding and IL-2. Crosslinking the cell-bound CD3 antibodies by plastic bound goat anti-mouse antibodies activated both T-cell subsets optimally and increased the IL-2 production of the IgG1-CD3 stimulated cultures. The data show that T cells (T8 greater than T4) can be stimulated by CD3 antibody binding and IL-2, but that crosslinking the cell-bound CD3 antibodies is crucial for optimal T4 cell stimulation and IL-2 production.     

Monoclonal antibodies directed to different epitopes in the CD3-TCR complex induce different states of competence in resting human T cells.

After the initial stages of activation, T cells are not able to proliferate on their own but become competent to proliferate in response to exogenously added lymphokines. In the present study we compared the capacity of mAb directed to CD3 (OKT3, Leu4, UCHT1) or to common epitopes on the alpha/beta T-cell receptor (BMA 031, BMA 032) to induce competence in purified resting T cells. Stimulation with either soluble anti-CD3 or anti-alpha/beta TCR mAb rendered cells competent to progress to DNA synthesis in response to exogenous IL-2. In contrast, only soluble BMA 031 and BMA 032 were able to induce responsiveness to IL-4; anti-CD3 mAb had either to be immobilized or used in combination with anti-CD28 mAb to induce responsiveness to IL-4. Further, BMA 031-induced IL-4 responsiveness was selectively found in the CD45RA+ T cell subset. Analysis of early activation events revealed that the capacity of soluble BMA 031 and BMA 032 to induce responsiveness to IL-4 did not correlate with the ability of these mAb to increase the level of cytosolic Ca2+ or to induce detectable tyrosine phosphorylation. On the other hand, soluble Leu4 (anti-CD3) triggered an increase in both intracellular Ca2+ and tyrosine phosphorylation but was unable to induce IL-4 responsiveness. These data indicate that the induction of IL-2 and IL-4 responsiveness requires different sets of activation signals which can be induced by stimulating different epitopes in the CD3-TCR complex. This supports the concept that distinct activation pathways are coupled to the CD3-TCR complex.     

Activation of human thymocytes by antibodies to the CD3/T-cell receptor complex: triggering of different epitopes results in different signals.

Monoclonal antibodies (mAb) against the CD3/T cell receptor (TcR) complex were analyzed for their ability to activate human thymocytes. In addition to mAb detecting epitopes on the CD3 complex (OKT3, BMA 030) the activation potential of recently developed mAb against common epitopes on the alpha/beta T-cell receptor (anti-TcR mAb: BMA 031, BMA 032) was evaluated. Several differences were observed between the two types of mAb: (a) Binding of the tested anti-CD3 mAb to thymocytes resulted in a rapid increase in the level of cytoplasmic free calcium ions [Ca2+]i, whereas no significant changes in [Ca2+]i were detected in thymocytes stimulated with BMA 031 or BMA 032. (b) Induction of effective proliferation induced by mAb OKT3 depended on exogenous IL-2 and in addition on the presence of accessory cells or phorbol-ester. Proliferation induced by BMA 031 only required exogenous IL-2. (c) OKT3 but not BMA 031 inhibited proliferation of thymocytes induced via the CD2 molecule. These studies indicate that anti-CD3 and anti-TcR mAb transduce different signals in thymocytes. Since the two types of mAb are directed to the same molecular complex the observed differences also support the idea that there are functionally different compartments in the CD3/TcR complex which may activate different signaling pathways.     

Further characterization of cytotoxic T cells generated by short-term culture of human peripheral blood lymphocytes with interleukin-2 and anti-CD3 mAb

 In this study we have specifically investigated the participation of T cells in the cytotoxic activity of peripheral blood lymphocytes (PBL) activated by interleukin-2 (IL-2, 50 U/ml) alone or in combination with an anti-CD3 mAb (BMA030, 10 ng/ml, IgG2a). Purified CD3⁺ T cells, incubated in the presence of the anti-CD3 mAb for 4 days, mediated a cytotoxic activity against HL60 and U937 tumor cell lines. Several findings suggested the involvement of a redirected-cytotoxicity phenomenon, since the lytic process was restricted to target cell lines bearing the high-affinity Fcγ receptor (FcγRI) and T lymphocytes stimulated by IL-2 alone did not lyse these cell lines. Furthermore, anti-CD3 mAb F(ab′)₂, anti-CD3 IgG1 (UCHT1), phytohemagglutinin or staphylococcal enterotoxin A did not induce a similar cytotoxic activity in T lymphocytes. The cytotoxic process occurred in the presence of a very low level of anti-CD3 antibodies (in the nanomolar range). The cytotoxic activity of T cells stimulated by IL-2 or by IL-2 + BMA030, against OVCAR-3 cells (MOv18⁺ ovarian tumor cell line), was also compared in the presence of a bispecific antibody (OC/TR, anti-CD3 × MOv18). The stimulation by IL-2 + BMA030 induced approximately a twofold higher cytotoxic activity than IL-2-activated T cells. This could be related to the state of activation of effector cells stimulated by IL-2 + BMA030, since the phenotypic analysis showed an increased proportion of T cells expressing several activation/differentiation markers (CD25, HLA-DR, CD45R0, adhesion molecules). These findings could be applied to the design of therapeutic protocols using anti-CD3 ×antitumoral bispecific antibodies. Received: 6 December 1995 / Accepted: 4 June 1996     

Reversal of CD45R isoform switching in CD8+ T cells.

Both CD4+ and CD8+ T cells express either CD45RA or CD45R0 isoform of CD45R in an exclusive way. Recent reports have shown that CD45RA+ T cells lose CD45RA and gain CD45R0 upon activation. This switching has been suggested to be irreversible although more recently, examples of reversal of CD45R isotype switching in CD4+ T cells have been reported. We report here that freshly isolated unprimed CD8+ T cells, when activated with PHA, temporarily lose CD45RA but reexpress an intermediate level of CD45RA 2-3 weeks after activation with PHA. This reversal seems to take place much more slowly in unprimed CD4+ T cells: the majority of CD4+ T cells that had lost CD45RA and gained CD45R0 remained CD45RA-CD45R0+ in 3 weeks after the stimulation. Also, long-term CD8+ CD45RA+ T cell lines stimulated with PHA or OKT3 showed even more rapid recovery of CD45RA while PPD-specific CD4+ T cell clones retained the original CD45R0 phenotype 3 weeks after stimulation with PPD or PHA.     

Efficiency of T cell triggering by anti-CD3 monoclonal antibodies (mAb) with potential usefulness in bispecific mAb generation

 T cell triggering can be achieved by monoclonal antibodies (mAbs) specific for the CD3/TcR complex. In the presence of appropriate costimulation and/or progression factors, such triggering permits the generation of effector cells for immunotherapy protocols involving the redirection of T cell lysis against tumor cells by mAbs bispecific for anti-CD3/anti-tumor cells (bs-mAbs). Focusing our analysis on the clinically relevant bs-mAb OC/TR, we found that bs-mAbs generated with the same anti tumor specificity, but two other anti-CD3 mAbs, TR66 and OKT3, have the same and a significantly lower lytic potential, respectively, compared with that of OC/TR. To evaluate the relevance of the anti-CD3 component, we examined several anti-CD3 mAbs with respect to binding parameters and the ability to trigger T lymphocytes. Competitive binding assays suggested that all anti-CD3 mAbs recognized the same or overlapping epitopes, although mAbs BMA030 and OC/TR bound with lower avidity than did αCD3 (the bivalent anti-CD3 mAb produced by the hybrid hybridoma OC/TR), TR66 and OKT3, as determined by measurement of the affinity constants. In all lymphocyte populations examined, which included resting peripheral blood mononuclear cells (PBMC), activated PBMC and T cell clones, OKT3, BMA033 and OC/TR failed to mobilize Ca²⁺ without cross-linking, whereas αCD3, in both murine and murine-human chimeric versions, TR66 and BMA030, did not require cross-linking. The ability to induce CD3 modulation was associated in part with the induction of Ca²⁺ fluxes. Despite the differences in the behavior of these mAbs in triggering the events that precede proliferation, all of them ultimately led to expression of the IL-2 receptor and to proliferation in T cells in the presence of accessory cells. Our data suggest that anti-CD3 mAbs that bind more rapidly (strong Ca²⁺ mobilizers) and more tightly under physiological conditions are good candidates for retargeting T cells in the bs-mAb clinical application. Received: 2 January 1997 / Accepted: 6 February 1997     

Activation of resting T lymphocytes by anti-CD3 (T3) antibodies in the absence of monocytes

The antigen receptor molecules on human T lymphocytes are noncovalently associated on the cell surface with the CD3 (T3) molecular complex. Perturbation of this complex with anti-CD3 monoclonal antibodies induces T cell activation. Previous studies have demonstrated that this process requires the participation of monocytes. In the present report, we demonstrate that purified, resting (G0 phase) T cells incubated with monoclonal anti-CD3 antibodies proliferate in response to purified interleukin 2 (IL 2), in a lymphokine dose-dependent fashion. Anti-CD3 antibody or IL 2 alone did not trigger cell division. The effect was specific for anti-CD3 antibodies because monoclonal antibodies reactive with other surface molecules (OKT4, OKT8, L368) were inactive. Furthermore, the same phenomenon was observed when anti-CD3 antibody Leu-4 (IgG1) was incubated with cells of individuals whose monocytes cannot process antibodies of the IgG1 subclass (Leu-4 nonresponders). In addition, both F(ab')2 and Fab fragments of anti-CD3 antibody OKT3 were also capable of rendering T cells receptive to the IL 2 growth signal. These data indicate that neither monocytes nor CD3 receptor cross-linking are required absolutely for resting T cell activation, provided that IL 2 is supplied exogenously. T lymphocytes treated with anti-CD3 antibodies proliferated in response to both purified mitogen-induced and recombinant IL 2. Antibodies to the IL 2 receptor (anti-Tac) inhibited the proliferation. Thus, the most likely mechanism for anti-CD3 antibody-mediated triggering is induction of IL 2 receptors.     

Dichotomous effect of monocyte Fc receptor interaction on anti-CD3-induced immunoglobulin synthesis

Monoclonal antibodies against the TCR/CD3 complex are capable of activating T cells which in turn may induce immunoglobulin synthesis in B cells under appropriate conditions. Here we present evidence that distinct immune responses, induced by four commonly used TCR/CD3 mAb (Leu4, OKT3, BMA030, BMA031) were related to the mAb interaction with monocyte Fc receptors for IgG. Depending on their isotype and on the technique by which they were crosslinked, TCR/CD3 mAb induced variable IgM and IgG synthesis in PBMC: If the mAb were crosslinked by monocyte IgG-Fc receptors they induced a high Ig production, while crosslinking the same mAb by plastic-bound goat anti-mouse antibodies (panning) failed to do so. Nevertheless, both crosslinking techniques triggered a strong proliferation and IL-2, IL-4, and IFN gamma lymphokine gene expression. The lack of Ig production under panning conditions was due to an additional IgG-Fc receptor interaction with monocytes: (a) If namely mAb F(ab')2 fragments, or mAb isotypes unable to bind to monocyte Fc receptors (IgG2b, IgG1 in nonresponders) were crosslinked by panning, both a good proliferation as well as Ig production ensued; (b) if TCR/CD3 mAb isotypes which could additionally bind to monocyte Fc receptor (IgG2a) were crosslinked, no Ig production occurred; (c) if mAb F(ab')2 fragments were crosslinked with a second anti-T cell antibody of IgG2a isotype, which could bind to monocyte Fc receptors, Ig synthesis was reduced. Interestingly enough, this diminishing effect, due to monocyte Fc receptor interaction, was only observed if CD4-positive cells were proliferating, but not if CD8-positive cells were activated.     

Age-related changes in the activation requirements of human CD4+ T-cell subsets.

In agreement with previous studies, we found that the proliferative response of unfractionated T-cells to phytohaemagglutinin (PHA) was severely impaired in healthy aged individuals (70-85 years). On the other hand, we did not observe significant differences between aged and young adults in T-cell responsiveness to mab OKT3 (anti-CD3). PHA responses in "old" T-cells could be substantially improved, however, by the addition of recombinant interleukin-2 (rIL-2) or KOLT2 (anti-CD28 mab). When individual CD4+ T-cell subpopulations were isolated from young and old donors and stimulated with PHA in the presence of autologous accessory cells, age-related deficiencies were seen in both CD4+CD45RA+ (naive) and CD4+CD45RO+ (memory) cell populations. Further analysis using a panel of coactivators in cultures depleted of accessory cells identified specific abnormalities in the CD2 or alternate pathway of T-cell activation. These were predominantly seen in CD4+ naive T-cells. The capacity of rIL-2, KOLT2, and PMA to restore, at least partially, T-cell responsiveness in the aged suggests a defect(s) in an early signal transduction mechanism.     

IL-2-R beta expression and function within resting CD8+ T cells preferentially segregate with the CD45R0+ subset.

Human peripheral blood CD8+ T cells constitutively express a low level of IL-2-R beta chains which were shown in this study to be preferentially carried by the CD45R0+ subset. Such receptors can transduce signals for in vitro IL-2-induced cytolytic function and for the initiation of soluble anti-CD3 and IL-2-induced cell proliferation. Using these stimulation models, a comparison was made between the responsiveness of resting, small CD45R0+ and CD45RA+ subpopulations of CD8+ T cells, both of them being isolated by negative selection and rigorously depleted of monocytes and of IL-2-inducible non-MHC-restricted CTL. Strong proliferation was induced in CD8+/CD45R0+ cells in response to IL-2 and soluble anti-CD3 (each of these stimuli being by itself ineffective), while in contrast, CD8+/CD45RA+ cells manifested, in this system, little reactivity. Accordingly, no conversion to the CD45R0 phenotype occurred in single stained CD45RA+ T cells following their incubation with the stimuli. A similar restriction of reactivity to CD8+/CD45R0+ T cells was observed with respect to IL-2-induced targetable T cell cytotoxicity. The CTL activity induced by IL-2 alone occurred without cell division. In contrast, the additional increase in CTL activity occurring upon the synergistic actions of anti-CD3 mAb and IL-2 coincided with intense cell proliferation, with no generation of LAK activity. The inhibition exerted by anti-IL-2-R beta mAb in the cytolytic and the proliferative activities induced by these stimuli in resting CD8+/CD45R0+ T cells emphasizes the importance of constitutive IL-2-R beta chains in the biology of these cells.     

Sustained linked stimulation via CD3 and CD4 is required for the IL-4-independent development of IL-4 synthesizing CD4+ T cells

Previous work has shown that CD4 engagement can promote the development of interleukin-4-producing cells from naive CD4+ T cells activated with anti-CD3 antibody and interleukin-2 in the absence of other exogenous signals, including interleukin-4 itself. When CD44low CD4+ T cells were activated with immobilized anti-CD3 antibody and interleukin-2, they proliferated and produced interferon-gamma but not interleukin-4. Co-immobilization of antibodies to CD3 and CD4 enhanced cell recoveries and induced interleukin-4 as well as interferon-gamma synthesis. Here we show that these effects of CD4 ligation were not observed when anti-CD4 antibody was replaced with another CD4 ligand, interleukin-16, or when the anti-CD3 and anti-CD4 antibodies were spatially separated by immobilization on different beads. Removal of the anti-CD4 antibodies within the first three days of stimulation also prevented the development of detectable interleukin-4-producing cells. The data suggest that interleukin-4-independent priming of interleukin-4-producing cells in this system requires sustained stimulation via both the T cell receptor and CD4 with close physical association between the ligands for these two receptors.     

Identification of the anti-CD3-unresponsive subpopulation of CD4+, CD45RA+ peripheral T lymphocytes.

The majority of peripheral CD4+ T lymphocytes proliferate in vitro in response to anti-CD3 in presence of autologous APC. The present study describes a subpopulation of CD4+ T cells that cannot be activated and progress into cell cycle by stimulation with anti-CD3 plus APC or with mitogenic combinations of anti-CD2. The in vitro responses of these anti-CD3-unresponsive CD4+ T cells were investigated with a panel of mAb to CD2, CD3, and CD28, and found to be similar to those previously observed for mature thymocytes: only the combination of anti-CD2 plus anti-CD28 produced cell proliferation. Anti-CD3-unresponsive T cells were CD45RA+, but represented only 14 to 22% of the CD4+, CD45RA+ T cell population. Activation with anti-CD2 plus anti-CD28 mAb resulted in major changes in the cell surface phenotype and functional properties: a loss of CD45RA+ occurred and an increased expression of CD45RO, CD29, and CD58 (LFA3), as well as a gain in responsiveness to anti-CD3 and anti-CD2. This change in CD45 phenotype from CD45RA to CD45RO occurs in both the anti-CD3-responsive and in the anti-CD3-unresponsive subsets of the CD45RA+, CD4+ cells after cell proliferation. The anti-CD3-unresponsive subset may represent a pool of not yet fully differentiated peripheral T cells. The acquisition of anti-CD3 responsiveness could occur as a consequence of Ag priming or by an Ag-independent mechanism. Involvement of the CD28 Ag in this process is suggested from the present study.     

Differential activation of phosphotyrosine protein phosphatase activity in a murine T cell hybridoma by monoclonal antibodies to CD45.

The effect of two anti-CD45 (T200, LCA, Ly5) antibodies on the activation of the murine T-cell hybridoma 13.13 has been evaluated. These studies have been carried out in a system that did not require cross-linking or coclustering of antibodies. Activation of 13.13 cells with the anti-CD3 monoclonal antibody, 145.2C11, gave rise to rapid increases in intracellular calcium and interleukin-2 production. Additionally, within 1 min, phosphorylation on tyrosine of four major proteins of about 130,000, 110,000, 80,000, and 37,000 daltons could be seen. Pretreatment of the cells with the anti-CD45 mAb M1/89.18.7.HK markedly inhibited all three biological responses, while an alternate anti-CD45 antibody, M1/9.3.4.HL.2, had little effect. The two antibodies bound to CD45 with similar affinities, and no differences in the lateral mobility of antibody-CD45 complexes in the cell membrane were observed. The inhibition of activation of the cells by M1/89.18.7.HK was abrogated significantly both by the phosphotyrosine protein phosphatase inhibitor orthovanadate and by excess M1/9.3.4.HL.2. If M1/89.18.7.HK was added to the 13.13 cells after they had already been activated with anti-CD3, it very effectively stimulated dephosphorylation of substrates that had been phosphorylated on tyrosines prior to adding the anti-CD45 antibody. These results indicate that the phosphotyrosine protein phosphatase activity of CD45 is critical to its biological function and that bivalent (i.e. uncross-linked) anti-CD45 antibodies can give rise to markedly different responses. One of the antibodies, M1/89.18.7.HK, appears to behave much like a receptor ligand and is able to activate the enzymatic activity associated with the CD45 transmembrane protein.     

Human resting B lymphocytes can serve as accessory cells for anti-CD2-induced T cell activation.

Although resting B cells are poor accessory cells for signals transmitted through the TCR/CD3 complex, we report that these B cells can support T cell proliferation when T cell activating signals are delivered through CD2. This was first suggested when leucine methyl ester treatment of PBMC abolished proliferation induced by anti-CD3, but not by the accessory cell-dependent anti-CD2 mAb combination, GT2 and OKT11. Then we demonstrated that unstimulated, resting B cells could support the proliferation of both CD4+ and CD8+ T cells. Aggregated IgG inhibited proliferation, suggesting that anti-CD2 mAb bound to T cells were cross-linked by attachment to B cell FcR. Two lines of evidence suggested that lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 interaction was crucial for anti-CD2-induced proliferation. First, proliferation was blocked by mAb against these adhesion molecules. Second, intercellular adhesion molecule-1 expression rapidly increased on resting B cells after the addition of anti-CD2, but not anti-CD3. This was of interest because fixed monocytes, but not fixed B cells, were able to support the proliferative response. In contrast to lymphocyte function-associated Ag-1/intercellular adhesion molecule-1, CD28/B7 interaction was not required for anti-CD2-induced proliferation, although ligation of these molecules provided important costimulatory signals for stimulation by anti-CD3. Finally, neutralizing antibodies against IL-1 alpha, IL-1 beta, and IL-6 showed only modest inhibitory effects on T cell proliferation. The addition of IL-1 and/or IL-6 to T cells failed to substitute for accessory cells and were only partially effective with fixed B cells. Further evidence of a linkage between CD2 and CD45 isoforms was obtained. Anti-CD45RA, but not anti-CD45RO, potentiated anti-CD2-induced T cell proliferation. These studies have revealed a novel role for resting B cells as accessory cells and have documented costimulatory signals that are important for this effect. Because Ag-presentation by resting B cells to T cells generally leads to T cell nonresponsiveness, it is possible that this tolerogenic signal may be converted to an activation signal if there is concurrent perturbation of CD2 on T cells.     

Stimulation of human T cells via anti-T cell receptor monoclonal antibody BMA031: distinct cellular events involving interleukin-2 receptor and lymphocyte function antigen 1

We have analyzed activation of resting human T cells by anti-T cell receptor (TCR) monoclonal antibody (mAb) BMA031, a murine mAb of the G2b isotype. Human peripheral blood lymphocytes (PBL) respond to anti-TCR mAb by short-term proliferation in vitro and by acquisition of responsiveness to interleukin 2 (rIL-2) in the absence of detectable IL-2 production. Cell depletion and limiting dilution experiments indicate that anti-TCR mAb +/- rIL-2 stimulation covers a substantial portion of human T cells, including CD4+ and CD8+ cells. Enhancement by rIL-2 of anti-TCR mAb-induced proliferation is blocked by anti-IL-2 receptor (IL-2R, p55) mAb, while anti-TCR mAb-induced proliferation is not. In contrast, anti-TCR mAb-induced proliferation is blocked by anti-lymphocyte function antigen 1 (LFA-1, CD11a) mAb and is not demonstrable in PBL from two patients with severe congenital LFA-1 deficiency, not even in the presence of irradiated LFA-1+ PBL. We conclude that stimulation of resting human T cells by anti-TCR mAb BMA031 enables dissociation of distinct steps in T cell activation that specifically require participation of IL-2R (p55) and LFA-1 cell surface molecules in a mutually exclusive way.     

A major myristylated substrate of protein kinase C and protein kinase C itself are differentially regulated during murine B- and T-lymphocyte development and activation.

The regulation and expression of protein kinase C (PKC) and phosphomyristin C (PMC) (a principal substrate of PKC which is the major myristylated protein in lymphocyte and glioma lines that express it) in murine B and T lymphocytes were investigated. Both PMC and PKC are differentially regulated during T-cell development. The level of PMC expression is highest in CD4-8-, intermediate in CD4+8+, and lowest in J11d-, CD4, or CD8 single-positive thymocytes. PKC is equally expressed by all three thymic populations. In striking contrast to thymocytes, resting peripheral lymph node T cells and T-cell clones express little if any PMC and reduced levels of PKC. Neither PKC nor PMC is significantly induced upon the activation of lymph node T cells: treatment with anti-CD3 antibodies or anti-CD3 and interleukin-2 fails to induce PKC, whereas PMC is not induced by anti-CD3 alone and is only slightly induced by anti-CD3 and interleukin-2. In contrast to the situation with T cells, PMC and PKC are constitutively expressed at moderate levels in mature B cells. PMC is greatly increased in B-cell blasts generated by cross-linking the antigen receptor with anti-immunoglobulin. These results demonstrate that PMC and PKC are differentially regulated during the development and activation of B and T cells, suggesting that cellular events that rely upon PKC and PMC may differ during ontogeny and activation of different lymphocyte subsets.     

CD27, a member of the nerve growth factor receptor family, is preferentially expressed on CD45RA+ CD4 T cell clones and involved in distinct immunoregulatory functions.

CD27 is a disulfide-linked 120-kDa homodimer expressed on the majority of peripheral T cells at variable density that belongs to the recently defined nerve growth factor receptor family. mAb reactive with CD27 can either enhance or inhibit T cell activation, suggesting a crucial role in the process of T cell activation. We now show that CD27 is preferentially expressed on the CD45RA+CD45RO-CD29low subset of CD4 cells. CD27 expression on this subset is maintained for a prolonged period in culture after PHA activation. In contrast, CD45RA-CD45RO(+)-CD29high subset of CD4 cells express very low level of CD27, and its expression is lost within 2 wk after PHA activation. To further analyze the differential expression of CD27 on these reciprocal subsets of CD4 cells, we developed T cell clones by stimulating isolated CD4+CD45RA+ and CD4+CD45RO+ populations with PHA. T cell clones derived from cells originally CD45RA+ retained both CD45RA and CD27 expression, whereas T cell clones derived from cells originally CD45RO+ were CD45RA- and CD27-. In functional assays, IL-4 production could only be induced in CD45RA-CD27- CD4 clones by stimulation with PMA and ionomycin. Four of six CD45RA+ CD4 clones had suppressor activity in PWM-driven IgG synthesis, whereas five of six CD45RA- CD4 clones had helper activity. Of interest, the suppressor activity of CD45RA+CD27+ clones was partially blocked by pretreatment with anti-CD27 mAb (1A4). Anti-1A4 pretreatment of these T cell clones resulted in elevation of intracellular cAMP levels. Thus, CD27 appears to play a role in the function of CD45RA+CD27+ CD4 cells, and may be involved in suppressor activity of these cells at least in part via its effects on cAMP production.     

Construction, expression and characterization of humanized antibodies directed against the human alpha/beta T cell receptor.

Completely humanized antibodies with specificity for the human alpha/beta TCR have been produced by genetic engineering. The L and H chain V region exons encoding the murine mAb BMA 031 CD regions and human EU framework regions were synthesized and replaced into previously isolated genomic fragments. These fragments were inserted into mammalian expression vectors containing the human kappa and gamma 1 C region exons. Two variants were constructed each containing selected BMA 031 amino acids within the human frameworks. The humanized genes were transfected into Sp2/0 hybridoma cells by electroporation and transfectomas secreting humanized antibody were isolated. Levels of antibody expression up to 7 pg/cell/24 h were obtained. The humanized antibody, BMA 031-EUCIV2, competed poorly with murine BMA 031 for binding to T cells. BMA 031-EUCIV3, however, bound specifically to T cells and competed effectively with both the murine BMA 031 antibody and a previously constructed chimeric BMA 031 antibody for binding to these cells. The relative affinity of BMA 031-EUCIV3 was about 2.5 times lower than BMA 031. The ability to promote antibody dependent cell-mediated cytolysis was significantly enhanced with the engineered antibodies as compared to murine BMA 031. Humanized BMA 031 is a clinically relevant, genetically engineered antibody with potential uses in transplantation, graft vs host disease, and autoimmunity.     

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.