Crosslinking CD3 with CD2 using sepharose-immobilized antibodies enhances T lymphocyte proliferation

T lymphocyte proliferation can be triggered through interactions with either CD3:Ti, the target of antigen-specific activation, or CD2, the target of an antigen-independent activation pathway. Sepharose-immobilized antibody reactive with CD3 was used to aggregate the T cell receptor complex resulting in T lymphocyte activation. When CD3 was simultaneously crosslinked with CD2 using Sepharose beads coupled to antibodies directed at both determinants, T cell proliferation was markedly enhanced (stimulation index = 8- to 11-fold). A smaller enhancement was induced when CD3 was crosslinked with several other functionally relevant T cell surface molecules. The relative mitogenic potency of the accessory molecules tested was CD2 greater than CD4 greater than CD8 greater than 2H4. Little or no increased proliferation resulted from crosslinking CD3 with class I or class II major histocompatibility antigens. The added proliferation induced by CD3: CD2 crosslinking did not occur in the presence of soluble antibodies directed against CD2. Human thymocytes, the majority of which express both CD3 and CD2, were similarly activated by Sepharose-immobilized antibodies. Our results suggest that specific interactions between T cell surface molecules may play a role in the regulation of lymphocyte activation.     

The T11 (CD2) molecule is functionally linked to the T3/Ti T cell receptor in the majority of T cells

Most mature human T lymphocytes express both the multichain T3 (CD3)/Ti T cell receptor for antigen (TCR), and the biochemically distinct 55-kDa T11 (CD2) glycoprotein. Stimulating the T11 molecule causes profound T cell proliferation and functional activation in vitro, but the relationship of T11-mediated activation to antigenic stimulation of T lymphocytes in vivo remains unknown. We now present evidence that T11 function is directly linked to TCR components in T3/Ti+ T11+ human T cells. First, we found that stimulating peripheral blood T cells with the mitogenic combination of anti-T11(2) cells with the mitogenic combination of anti-T11(2) plus anti-T11(3) monoclonal antibodies caused the phosphorylation of TCR T3 chains. The predominance of T3-gamma-phosphorylation that occurred in anti-T11(2) plus anti-T11(3)-treated T cells is similar to the pattern previously observed in antigen-stimulated T cell clones. Second, T11 function depended upon concurrent cell-surface expression of the TCR. Thus, when peripheral blood T cells were deprived of cell surface T3/Ti by anti-T3 modulation, anti-T11(2) plus anti-T11(3)-induced mitogenesis and transmembrane signal generation in the form of calcium mobilization were inhibited. The mechanism of TCR-T11 interdependence was investigated in a series of TCR-deficient variants of a T cell lymphoblastoid cell line. T3/Ti negative variants expressed cell surface T11, but anti-T11(2) plus anti-T11(3) failed to cause detectable calcium mobilization. The TCR-deficient variants also failed to express T11(3) activation epitopes after incubation with anti-T11(2) antibodies, suggesting that T11(3) expression is an essential and TCR-dependent intermediate in the T11 activation mechanism in these cells. Taken together, our results suggest that T11 function depends upon cell-surface expression of TCR in many T3/Ti+ T11+ T lymphocytes, and T11-mediated activation is intimately interconnected with TCR activation mechanisms. A model in which stimulating signals delivered via T11 may be a part of antigenic activation of T lymphocytes is presented.     

Heterogeneity of human T4+ inducer T cells defined by a monoclonal antibody that delineates two functional subpopulations

A monoclonal antibody termed anti-T4 that detected approximately 60% of peripheral blood T lymphocytes was shown to define the human inducer population. In the present study, we characterized three additional monoclonal antibodies, anti-T4A, anti-T4B, and anti-TQ1, that were reactive with a similar percentage of T lymphocytes. Anti-T4A, anti-T4B, and anti-T4 delineated identical cell populations, while those defined by anti-TQ1 differed in several respects: 1) Anti-TQ1 stained a minority (less than 7%) of thymocytes, whereas the other antibodies stained a majority (80%); 2) Anti-TQ1 reacted with 70 to 85% of T4+ lymphocytes, but also stained 50% of T cells within the T4- (T8+) cytotoxic/suppressor subset; 3) The antigen defined by anti-TQ1 was not restricted in its expression to T cells; it defined a fraction of normal B and null lymphocytes as well as non-T cell lines. In vitro studies indicated that the subpopulations of T4+ T lymphocytes delineated by anti-TQ1 were functionally distinct. Although T4+TQ1+ and T4+TQ1- T cells proliferated in an equal fashion to soluble antigen and alloantigen, only the T4+TQ1+ subset was responsible for maximal proliferation in autologous MLR. This T4+TQ1+ subset contained a population of lymphocytes reactive with the previously defined JRA autoantibody. In contrast, the T4+TQ1-, but not the T4+TQ1+, subset provided the majority of T cell help for B cell immunoglobulin production in a pokeweed-driven system. We conclude that the subpopulation of T4+ inducer cells responsible for maximal helper activity in T-B interactions is restricted to a minor subpopulation of T4+ lymphocytes.     

Antibodies to the L3T4 and Lyt-2 molecules interfere with antigen receptor-driven activation of cloned murine T cells

When L3T4+ cloned murine helper T lymphocytes (HTL) are stimulated with antigen or immobilized anti-T cell receptor (TCR) monoclonal antibodies (mAb) at concentrations which are optimal for proliferation, anti-L3T4 mAb inhibits activation as measured by proliferation and lymphokine production. Under similar conditions, IL 2-independent proliferation of Lyt-2+ cloned murine cytolytic T lymphocytes (CTL) stimulated by anti-TCR mAb is inhibited by anti-Lyt-2 antibodies. Proliferation of cloned HTL and CTL cells stimulated by IL 2 is not affected by the anti-L3T4 and anti-Lyt-2 mAb. The inhibition of TCR-induced activation of the T cell clones is not due to interference with the binding of the anti-TCR mAb. Stimulation of the TCR has been proposed to induce lymphokine secretion and proliferation by T cells through a pathway involving the activation of protein kinase C and the stimulation of an increase in the concentration of intracellular free calcium. However, proliferation of T cells stimulated by PMA (which activates protein kinase C) plus the calcium ionophore A23187 (which increases the concentration of intracellular free calcium) is not affected by mAb reactive with the Lyt-2 or L3T4 structures. If TCR stimulation does indeed activate T cells by activating protein kinase and increasing intracellular free calcium, then our data suggest that anti-L3T4 and anti-Lyt-2 mAb inhibit TCR-driven proliferation at some step before the activation of protein kinase C and the stimulation of a rise in intracellular free calcium concentration. Our results suggest that anti-L3T4 and anti-Lyt-2 mAb interfere with early biochemical processes induced by stimulation of the TCR. In HTL, which proliferate via an autocrine pathway, anti-L3T4 mAb appears to inhibit proliferation by interfering with signaling events involved in lymphokine production. Inhibition of IL 2-independent proliferation of Lyt-2+ cells by anti-Lyt-2 mAb appears to occur by a different mechanism. The precise molecular basis for the interference of each cell type has not yet been characterized.     

The T4 molecule differentially regulating the activation of subpopulations of T4+ cells

It has been demonstrated that the T4+2H4+ subset functioned as a suppressor inducer cell, whereas the reciprocal T4+2H4- subset provided help for B cell Ig production. In the present studies, a series of monoclonal antibodies to cell surface structures expressed on these subsets of cells were examined for their effects on the proliferative and immunoregulatory functions generated in AMLR. We demonstrated that anti-T4 antibody preferentially inhibited the proliferative response of the T4+2H4+ but not T4+2H4- cells against self-MHC antigens. In contrast, anti-T3 and anti-Ia antibodies inhibited the response of both subsets of cells. This subset preference of anti-T4 antibody was not attributable to either the isolation procedures used or a shift in the kinetics of proliferation to autologous self-MHC antigens. Moreover, both IL 2 production and the immunoregulatory function of the T4+2H4+ subset was profoundly inhibited by anti-T4 antibody, whereas the T4+2H4- subset was minimally influenced. In the absence of Ia molecules, T4+2H4+ but not T4+2H4- cell proliferation was inhibited with anti-T4 antibody. Together, these results suggest that the T4 molecule plays a distinct functional role in the differential triggering of subsets of T4+ cells.     

Human epidermal cells from ultraviolet light-exposed skin preferentially activate autoreactive CD4+2H4+ suppressor-inducer lymphocytes and CD8+ suppressor/cytotoxic lymphocytes

In vivo exposure of human epidermis to UV abrogates the function of T6+DR+ Langerhans cells and induces the appearance of Ag-presenting T6-DR+ OKM5+ cells in the epidermis. Since UV exposure of murine skin results in Ts lymphocyte activation, we investigated the capacity of human epidermal cells (EC) harvested 3 days after in vivo UV exposure to activate regulatory and effector autologous T lymphocyte subsets. T lymphocytes were separated into CD8+ suppressor/cytotoxic lymphocytes and CD4+ helper/inducer lymphocytes by C lysis and panning. The CD4+ subset was further divided by using the 2H4 mAB to obtain CD4+2H4+ lymphocytes (inducers of TS lymphocytes) and CD4+2H4- lymphocytes (inducers of B cell Ig production and inducers of cytotoxic T cells). Unirradiated suction blister-derived EC from control skin (C-EC) and from skin exposed in vivo to UV (UV-EC) were cultured with purified autologous T lymphocyte subsets in the absence of added Ag. The resultant T lymphocyte proliferation was detected by [3H]thymidine uptake. UV-EC were highly effective in the stimulation of CD4+ lymphocytes, whereas C-EC were poor stimulators. The stimulator effect of UV-EC was abrogated after depletion of DR+ UV-EC. When CD4+ lymphocytes were fractionated, UV-EC consistently demonstrated enhanced ability to stimulate suppressor-inducer CD4+2H4+ lymphocytes relative to C-EC. Although less responsive than CD4+2H4+ lymphocytes, CD4+2H4- lymphocytes also demonstrated greater proliferation to UV-EC than to C-EC. Neither UV-EC nor C-EC were able to activate CD8+ lymphocytes devoid of CD4+ lymphocytes. However, after addition of rIL-2 at concentrations that allow binding only to the high affinity IL-2R on T lymphocytes, UV-EC induced vigorous proliferation of CD8+ lymphocytes, whereas C-EC induced only background levels of proliferation. C lysis of leukocytes resident within UV-EC resulted in 66 to 70% reduction of CD8+ lymphocyte proliferation. In conclusion, UV-EC may activate CD8+ lymphocytes by at least two pathways: (1) UV-EC activation of CD4+2H4+ lymphocytes may induce differentiation/proliferation of CD8+ suppressor cells and (2) UV-EC activation of CD4+ cells may induce IL-2 production, that, in combination with UV-induced epidermal leukocytes, stimulates CD8+ cells.     

Comodulation of CD3 and CD4. Evidence for a specific association between CD4 and approximately 5% of the CD3:T cell receptor complexes on helper T lymphocytes

The aggregation of a specific class of lymphocyte surface molecules results in patching, capping, and surface modulation of the aggregated ligand. Both CD4, an associative recognition structure found on helper T lymphocytes, and CD3, a component of the T cell receptor complex, are members of this functional subgroup. When 125I-labeled monoclonal antibodies reactive with either CD4 (19Thy 5D7) or CD3 (RW24B6) were bound to T lymphocytes, the subsequent addition of goat anti-mouse Ig resulted in their rapid, temperature-dependent internalization. Whereas the binding of 125I-19Thy 5D7 (anti-CD4) was inhibited by greater than 90% in the presence of unlabeled 19Thy 5D7, no inhibition occurred in the presence of unlabeled antibody reactive with CD3 (RW28C8). We took advantage of the fact that these antibodies were of different isotypes (19Thy 5D7:IgG2a; RW28C8:IgGl) to determine whether the internalization of CD3 induced the comodulation of CD4. T lymphocytes preincubated with 125I-19Thy5D7 (anti-CD4) and unlabeled RA28C8 (anti-CD3) were treated with goat anti-mouse IgGl under conditions shown to quantitatively internalize CD3. After 1 h at 37 degrees C, T lymphocytes had internalized 10.5 +/- 2.6% (n = 3) of their antibody-bound cell surface CD4. After similar incubations with media alone or with goat anti-mouse IgGl in the absence of prebound RW28C8 (anti-CD3), no internalization of CD4 could be detected. Control antibodies reactive with CD45R (2H4, IgGl) also failed to induce the internalization of CD4. Similar results were obtained by using a helper T cell clone (T4C1) that internalized 9.6 +/- 2.8% (n = 3) of its antibody-bound cell surface CD4 in response to CD3 modulation. In a reciprocal experiment, 125I-anti-CD3 (RW24B6, IgG2b) was preincubated with T4Cl cells together with unlabeled anti-CD4 (12T4D11, IgG1) prior to the addition of goat anti-mouse IgGl. The quantitative modulation of CD4 induced the co-internalization of 4.6 +/- 0.6% (n = 3) of cell surface CD3. These results suggest that approximately 5% of the CD3:T cell receptor complexes on helper T lymphocytes are specifically associated with CD4. Furthermore, our results suggest that an average of two CD4 molecules associate with each CD3:T cell receptor complex.     

Trispecific F(ab')3 derivatives that use cooperative signaling via the TCR/CD3 complex and CD2 to activate and redirect resting cytotoxic T cells.

To investigate whether the retargeting of resting CTL can benefit from cooperative signaling between the TCR/CD3 complex and various accessory molecules, such as CD2, CD4, CD5, and CD8, we have constructed a series of trispecific F(ab')3 derivatives. Each derivative was designed to engage effector T lymphocytes with two Fab' arms, and tumor cells with a single Fab' arm. They were constructed by selective coupling of three mAb Fab' fragments, primarily via their hinge-region sulfhydryl groups, using the cross-linker o-phenylenedimaleimide. En route to the production of trispecific F(ab')3 antibodies a range of bispecific F(ab')2 derivatives was first prepared which could bind simultaneously to two different receptor molecules on T cells. Bispecific derivatives containing specificities for (CD2 (T11(1)) x CD3), (CD3 x CD4), (CD3 x CD8) or two epitopes on CD2, ((T11(1) x (T11(3)), all yielded two to three times the uptake of [3H]thymidine with fresh PBMC to that seen with intact IgG from anti-CD3 (OKT3). The exception to these findings was a bispecific F(ab')2 derivative with specificities for (CD3 x CD5) which caused slightly less proliferation than the control reagent, OKT3 IgG. When these bispecific antibodies were converted into trispecific antibodies (TsAb) by the addition of a Fab' from anti-CD37 they were then all able to retarget resting, unprimed, T cells from fresh PBMC for lysis of CD37+ tumor cells. However, the cytotoxic activity of these reagents fell into two distinct groups: group one, containing (anti-CD3 x anti-CD4 x anti-CD37), (anti-CD3 x anti-CD5 x anti-CD37), and (anti-CD3 x anti-CD8 x anti-CD37), gave minimal lysis and behaved in a similar way to the BsAb, (anti-CD3 x anti-CD37), i.e., no evidence of cooperative signaling for lysis; and group two, containing (anti-T11(1) x anti-CD3 x anti-CD37) and (anti-T11(1) x anti-T11(3) x anti-CD37), which were highly cytotoxic and gave up to 80% specific 51Cr-release. The failure of group one TsAb, in particular (anti-CD3 x anti-CD8 x anti-CD37) which should recruit CD8+ CTL, to give efficient lysis despite having anti-T cell arms that were mitogenic as a bispecific antibody, indicates that the cooperative signaling for proliferation is probably distinct from the signal(s) provided by group two TsAb that activate for both proliferation and lysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulation via the CD3 and CD28 molecules induces responsiveness to IL-4 in CD4+CD29+CD45R- memory T lymphocytes

Although both IL-2 and IL-4 can promote the growth of activated T cells, IL-4 appears to selectively promote the growth of those helper/inducer and cytolytic T cells which have been activated via their CD3/TCR complex. The present study examines the participation of CD28 and certain other T cell-surface molecules in inducing T cell responsiveness to IL-4. Purified small high density T cells were cultured in the absence of accessory cells with various soluble anti-human T cell mAb with or without soluble anti-CD3 mAb and their responsiveness to IL-4 was studied. None of the soluble anti-T cell mAb alone was able to induce T cell proliferation in response to IL-4. A combination of soluble anti-CD3 with anti-CD28 mAb but not with mAb directed at the CD2, CD5, CD7, CD11a/CD18, or class I MHC molecules induced T cell proliferation in response to IL-4. Anti-CD2 and anti-CD5 mAb enhanced and anti-CD18 mAb inhibited this anti-CD3 + anti-CD28 mAb-induced T cell response to IL-4. In addition, anti-CD2 in combination with anti-CD3 and anti-CD28 mAb induced modest levels of T cell proliferation even in the absence of exogenous cytokines. IL-1, IL-6, and TNF were each unable to replace either anti-CD3 or anti-CD28 mAb in the induction of T cell responsiveness to IL-4, but both IL-1 and TNF enhanced this response. The anti-CD3 + anti-CD28 mAb-induced response to IL-4 was exhibited only by cells within the CD4+CD29+CD45R- memory T subpopulation, and not by CD8+ or CD4+CD45R+ naive T cells. When individually cross-linked with goat anti-mouse IgG antibody immobilized on plastic surface, only anti-CD3 and anti-CD28 mAb were able to induce T cell proliferation. These results indicate that the CD3 and CD28 molecules play a crucial role in inducing T cell responsiveness to IL-4 and that the CD2, CD5, and CD11a/CD18 molecules influence this process.     

Amplification of suppressor inducer pathway with monoclonal antibody, anti-2H4, identifying a novel epitope of the common leukocyte antigen/T200 antigen

The 2H4 antigen, comprised of a 200/220-kDa glycoprotein of the leukocyte common antigen (LCA) family, is expressed on a suppressor inducer, but not a helper inducer subset of T4 cells. Earlier studies have demonstrated that the T4+2H4+ subset of cells maximally responded to the AMLR and this molecule has an important role in generated suppressor signals in AMLR/Con A-activated T cell systems. In the present study, we examined the effect of a series of monoclonal antibodies including anti-2H4 antibody on the initial activation of T4 cells in response to self-Ia antigens. We found that the addition of anti-2H4 antibody resulted in an augmentation of the proliferative response of T4 cells in AMLR, whereas other antibodies reactive with LCA/T200 antigens lacked this ability. Furthermore, anti-2H4 antibody enhanced both IL-2 production and IL-2R expression in this AMLR system. This enhancing effect was inhibited by anti-T3 antibody. Moreover, the suppressor inducer function of AMLR T4 cells was enhanced with anti-2H4 antibody by increasing the number of 2H4+ cells with high antigen density. Taken together, these results suggest that the 2H4 antigen may serve as an accessory structure for enhancing the activation of the T4+2H4+ suppressor inducer subset at initiation of cell triggering.     

Regulation of CD4 and CD8 surface expression on human thymocyte subpopulations by triggering through CD2 and the CD3-T cell receptor

Human thymocytes bearing the CD4 and/or CD8 antigens can be fractionated into cells with an immature and more mature phenotype based on their quantitative expression of the CD3 Ag (J. Immunol. 138:3108; J. Immunol. 139:1065). We show that the expression of CD4 and CD8 on thymocyte subpopulations with low CD3 (CD3L) and high CD3 (CD3H) is regulated by activation through the CD2 molecule and perturbation of the CD3-T cell receptor complex (CD3-Ti). Similar to its previously reported effects on peripheral T cells, PMA was able to induce the down-regulation of surface CD4, but not CD8, on thymocyte subpopulations. PMA could induce CD4 and CD8 phosphorylation in both CD3L and CD3H fractions. These results suggest that if changes in phosphorylation represent the mechanism by which CD4 and CD8 are able to transmit signals, this mechanism is operative in both CD3L and CD3H subpopulations. Treatment with anti-T11(2) and anti-T11(3) antibodies (CD2 activation pathway) resulted in partial down-regulation of CD4 but not CD8 surface expression on both CD3L and CD3H thymocytes. Similar treatment had no detectable effect on peripheral T cells. The down-regulation of surface CD4 induced by activation via CD2 could be inhibited by treatment of thymocytes with anti-CD3 antibodies. Treatment of thymocytes with anti-CD3 alone or following CD2 activation induced the selective down-regulation of surface CD8 within 15 minutes. These results suggest that CD2 and CD3-Ti triggering may regulate CD4 and CD8 surface expression on thymocytes. Furthermore, these results suggest that "cross-talk" between the CD2 and CD3-Ti pathway of activation may involve CD4 and CD8 molecules.     

Activation requirements for normal T cells: accessory cell-dependent and -independent stimulation by anti-receptor antibodies

We have examined the requirements for the activation of normal T cells by two anti-T cell receptor antibody preparations, including a rabbit antiserum, R3497, which binds to all normal T cells, and a rat monoclonal antibody, KJ16-133, which binds to about 20% of T cells. The requirements for stimulation of T cells by both antibodies were similar. Soluble antibodies in the absence of accessory cells (AC) failed to induce either proliferation or the expression of IL 2 receptors, and the addition of either IL 2 or PMA failed to synergize with these soluble antibodies for an AC-independent proliferative response. Activation could only be achieved in the presence of Fc receptor-positive AC, although Fc receptor expression alone appeared not to be sufficient for AC activity because some Fc receptor-positive cells did not function in this capacity. Activation with anti-receptor antibody conjugated to Sepharose 4B beads could be demonstrated in the presence of some exogenous cofactors, such as IL 2 and PMA, but not in the presence of recombinant IL 1. When activation by soluble antibody plus AC was compared to activation by bead-conjugated antibody + recombinant IL 2, it was found that the former favored the stimulation of Lyt-2+ cells. The effects of the addition of anti-L3T4 monoclonal antibody was also examined in this system. Anti-L3T4 inhibited the response of L3T4+ cells when used in the presence of Ia+ as well as Ia- AC, and it also inhibited activation in a system in which KJ16-133 conjugated to Sepharose was used in the absence of AC. Because anti-L3T4 had an inhibitory effect in the presence of Ia- AC as well as in the absence of any AC, it is concluded that L3T4 does not necessarily function by interacting with Ia on the surface of AC, and may directly transmit down-regulatory signals when bound by anti-L3T4.     

Roscovitine Suppresses CD4+ T Cells and T Cell-Mediated Experimental Uveitis

Background

T cells are essential for the development of uveitis and other autoimmune diseases. After initial activation, CD4+ lymphocytes express the co-stimulatory molecule OX40 that plays an important role in T cell proliferation. Cyclin dependent kinase 2 (CdK2) plays a pivotal role in the cell cycle transition from G1 to S phase. In addition, recent research has implicated CdK2 in T cell activation. Thus, we sought to test the immunosuppressive effect of roscovitine, a potent CdK2 inhibitor, on CD4+ T cell activation, proliferation, and function.

Design and Methods

Mouse CD4+ T cells were activated by anti-CD3 and anti-CD28 antibodies. The expression of OX40, CD44, and CdK2 were analyzed by flow cytometry. In addition, cell cycle progression and apoptosis of control and roscovitine-treated T lymphocytes were measured by BrdU incorporation and annexin V assay, respectively. Furthermore, the immunoregulatory effect of roscovitine was evaluated in both ovalbumin-induced uveitis and experimental autoimmune uveitis (EAU) models.

Results

In this study, we found that T cell activation induced OX40 expression. Cell cycle analysis showed that more CD4+OX40+ cells entered S phase than OX40- T cells. Concurrently, CD4+OX40+ cells had a higher level of CdK2 expression. Roscovitine treatment blocked activated CD4+ cells from entering S phase. In addition, roscovitine not only reduced the viability of CD4+ lymphocytes but also suppressed T cell activation and cytokine production. Finally, roscovitine significantly attenuated the severity of T cell-dependent, OX40-enhanced uveitis.

Conclusion

These results implicate CdK2 in OX40-augmented T cell response and expansion. Furthermore, this study suggests that roscovitine is a novel, promising, therapeutic agent for treating T cell-mediated diseases such as uveitis.     

Monoclonal antibodies to the human C3b/C4b receptor (CR1) enhance specific B cell differentiation

The addition of monoclonal antibodies against the human C3b/C4b receptor (CR1) to cultures of peripheral blood lymphocytes in the presence of suboptimal amounts of TNP bound to polyacrylamide beads enhanced by 150 to 400% the specific anti-TNP response, as measured by a plaque-forming cell assay on day 7. Anti-CR1 antibodies similarly enhanced the anti-fluorescein antibody response. Enhancement only occurred in cultures performed in the presence of the relevant antigen. No enhancing effect on the anti-TNP response was observed on addition to cultures of monoclonal antibodies directed against other surface antigens of B cells or an anti-T cell antibody of the same subclass as that of anti-CR1 antibodies. Anti-CR1 antibodies alone did not induce nonspecific B cell proliferation and did not provide B cells with a first signal for proliferation in the presence of a source of B cell growth factors. Anti-CR1 antibodies did not enhance the nonspecific proliferative response of B cells to growth factors derived from PHA-stimulated T cells, semi-purified BCGF 20 KD, BCGF 50 KD, or recombinant IL 2 in the presence of anti-mu. In this respect, the effect of anti-CR1 antibodies differs from that of anti-CR2 antibodies which interact with early stages of B cell activation. In contrast, anti-CR1 antibodies enhanced specific differentiation of antigen-activated B cells in the absence of T cells when soluble T cell factors were provided. Similar results were obtained by using either of two sources of differentiation factors, the MLA-144 supernatant or a 30 to 15 KD fraction from PHA-stimulated T cells. These results indicate that triggering of CR1 on B cells positively regulates the specific antibody response to low doses of antigen by enhancing B cell differentiation whether T cell help is provided by intact T cells or by T cell-derived differentiation factors.     

Cytochalasins enhance the proliferation of CD4 cells through the CD3-Ti antigen receptor complex or the CD2 molecule through an effect on early events of activation.

Cytochalasins are known to inhibit or enhance the proliferation of T cells induced by mitogens in a concentration-dependent fashion. To clarify the mechanism by which cytochalasins enhance T cell proliferation, we examined which activation pathways and events in signal transduction were affected by cytochalasins. We also examined subsets of CD4 cells for a preferential response to cytochalasins. Cytochalasins enhanced the proliferation of CD4 cells induced by optimal doses of anti-CD3 antibody or suboptimal doses of anti-CD2 antibodies. Cytochalasins, at low concentrations, enhanced the rise in intracellular Ca2+ and production of IP3 in CD4 cells activated by anti-CD2 or CD3 antibodies. Cytochalasins also enhanced the modulation of CD3 induced by anti-CD3 antibody. These results suggest that cytochalasins enhance the proliferation of CD4 cells by affecting early events in signal transduction after activation through the CD3-Ti Ag-receptor complex or CD2 molecule. At the doses used, cytochalasins appear to interact with cytochalasin-binding sites in the cell membrane. Cytochalasins predominantly enhanced CD3-mediated proliferation in the CD29-subset of CD4 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.     

Comitogenic effect of solid-phase immobilized anti-1F7 on human CD4 T cell activation via CD3 and CD2 pathways

We have recently developed a mAb, anti-1F7, which defines a family of structures found to include the molecule recognized by anti-Ta1 (CD26). In this paper, we demonstrated that binding of 1F7 by solid-phase immobilized anti-1F7 mAb but not anti-Ta1 mAb has a comitogenic effect by inducing proliferation of human CD4+ T lymphocytes in conjunction with submitogenic doses of anti-CD3 or anti-CD2. The proliferative response induced via the CD3-1F7 or CD2-1F7 pathways is associated with the IL-2 autocrine pathway, including IL-2 production. IL-2R expression and anti-IL-2R (Tac) inhibition. Furthermore, solid-phase immobilization of anti-1F7 but not anti-Ta1 acts in conjunction with submitogenic doses of PMA to mediate a comitogenic effect in the absence of anti-CD3 or anti-CD2, leading to CD4+ T cell proliferation. PMA treatment, in the meantime, leads to enhanced expression of 1F7 on the T cell surface. Despite its functional association with both pathways of activation, however, the 1F7 structure is not comodulated with the CD3/TCR complex nor the CD2 molecule. These findings thus suggest that the CD26 Ag is involved in CD3 and CD2-induced human CD4+ T cell activation.     

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.     

Engagement of CD28 modulates CXC chemokine receptor 4 surface expression in both resting and CD3-stimulated CD4+ T cells

Optimal CD4+ T cell activation requires the cooperation of multiple signaling pathways coupled to the TCR-CD3 complex and to the CD28 costimulatory molecule. In this study, we have investigated the expression of surface CXC chemokine receptor 4 (CXCR4) in enriched populations of CD4+ T PBL, stimulated with anti-CD3 and anti-CD28 mAbs, immobilized on plastic. Anti-CD3 alone induced a progressive down-regulation of surface CXCR4, accompanied by a significant decline in the entry of the HXB2 T cell line-tropic (X4-tropic) HIV-1 clone in CD4+ T cells. Of note, this effect was strictly dependent on the presence in culture of CD14+ monocytes. On the other hand, anti-CD28 alone induced a small but reproducible increase in the expression of surface CXCR4 as well as in the entry of HXB2 HIV-1 clone in resting CD4+ T cells. When the two mAbs were used in combination, anti-CD28 potently synergized with anti-CD3 in inducing the expression of CD69 activation marker and stimulating the proliferation of CD4+ T cells. On the other hand, anti-CD28 counteracted the CXCR4 down-modulation induced by anti-CD3. The latter effect was particularly evident when anti-CD28 was associated to suboptimal concentrations of anti-CD3. Because CXCR4 is the major coreceptor for the highly cytopathic X4-tropic HIV-1 strains, which preferentially replicate in proliferating CD4+ T cells, the ability of anti-CD28 to up-regulate the surface expression of CXCR4 in both resting and activated CD4+ T cells provides one relevant mechanism for the progression of HIV-1 disease.