Differences in surface phenotype between cytolytic and non-cytolytic CD4+ T cells. MHC class II-specific cytotoxic T lymphocytes lack Leu 8 antigen and express CD2 in high density

A number of cell surface molecules are differentially expressed on functionally distinct subsets of CD4+ T cells. However, to date CD4+ T cells capable of becoming CTL have not been shown to be phenotypically distinct from other CD4+ T cells, and in the current study we examined the ability of Leu 8+ and Leu 8- CD4+ subpopulations to become cytotoxic effectors after their stimulation with allogeneic lymphoblastoid cell lines. Although CD4+, Leu 8+ cells proliferated more vigorously than CD4+, Leu 8- cells in primary cultures stimulated with allogeneic LCL, the CD4+, Leu 8- population was the major source of cytotoxic effectors, killing targets with specificity for their class II MHC alloantigens. In most subjects, CD4+ precursors of CTL were distinguished not only by their lack of Leu 8 expression but also by their relatively high density of CD2, LFA-1, and LFA-3, molecules known to mediate non-specific cell-to-cell adhesion and postulated to be markers of immunologic memory. The absence of Leu 8 does not appear to be a reliable memory cell marker, however, because Leu 8+ as well as Leu 8-, CD4+ cells from PPD skin test positive subjects responded to the recall Ag, PPD. During 3 mo of continuous culture with allogeneic stimulators, Leu 8- cells retained their cytolytic activity and remained unreactive with anti-Leu 8 mAb, whereas Leu 8+ cells remained non-cytolytic and reactive with anti-Leu 8, suggesting that under the conditions used the Leu 8 phenotype is relatively stable. PHA or anti-CD3 mAb enhanced non-specific killing by alloantigen-stimulated CD4+,Leu 8- lines but failed to unmask any cytolytic potential in CD4+,Leu 8+ lines. We conclude that MHC class II-specific cytolytic CD4+ T cells can be distinguished from non-cytolytic CD4+ cells on the basis of their surface phenotype, and that most CD4+ CTL are contained within the Leu 8- subpopulation.     

Monoclonal antibody against the human peripheral lymph node homing receptor homologue (Leu 8) inhibits B cell differentiation but not B cell proliferation.

Previous studies have shown that a subpopulation of circulating human B cells expresses the Leu 8 peripheral lymph node homing receptor homologue and that these B cells are capable of producing Ig in response to staphylococcus A Cowan I (SAC). In the present study the effect of a signal delivered via the Leu 8 molecule (using anti-Leu 8 mAb) on B cells was examined. Initially, it was shown that immobilized anti-Leu 8 suppressed IgM and IgG secretion of B cells activated by SAC + IL-2 but not that by PWM-prestimulated B cells or B cells stimulated with PWM in the presence of CD4+, Leu 8- T cells (a source of helper cells). It was also shown that anti-Leu 8 did not suppress SAC + IL-2-stimulated B cell proliferation or expression of IL-2R alpha-chain or c-myc mRNA in B cells. The addition of T cells, monocytes, purified IL-2, rIL-1, rIL-6, or human B cell growth factor did not overcome the inhibitory effect of anti-Leu 8 on SAC-stimulated B cell Ig production, and the inhibitory effect of anti-Leu 8 was not blocked by anti-TGF-beta. Finally, inhibition of B cell differentiation occurred even when anti-Leu 8 was added up to 72 hrs after initiation of cell culture. Thus, anti-Leu 8 is unique among inhibitors of B cell function in that it can down-regulate immunoglobulin synthesis without affecting B cell proliferation. These findings suggest that a natural ligand for Leu 8 could affect not only homing of B cells, but also B cell differentiation.     

Two-dimensional flow cytometric analysis of T cell subsets maintained in IL-2 medium after autologous mixed leukocyte reaction activation

Human T cells are stimulated with an autologous mixed leukocyte reaction (AMLR) and can be propagated in interleukin-2. Staining of the cultured cells with the combination of two monoclonal antibodies was evaluated by two-dimensional flow cytometry at weekly intervals. AMLR activation resulted in an initial preservation of the CD4+ (helper/inducer T) subset predominance over the CD8+ (suppressor/cytotoxic T) cells, noted on normal circulating blood lymphocytes. However, during culture in interleukin-2, there was a progressive increase in the percentages of CD8+ Leu 15- cytotoxic T, CD4+ Leu 8- helper T, and CD3+ HLA-DR+ activated T cells, and a concomitant decrease in those of CD4+ Leu 8+ suppressor inducer T and CD8+ Leu 15+ suppressor T cells if the responder sheep red blood cell (SRBC)-rosetting T cells were made up by tris ammonium chloride, but not by hypotonic shock treatment to lyse SRBC. The significant difference between hypotonic shock-T cells and ammonium chloride-T cells in the phenotypic changes of T cell subsets after long-term culture in an interleukin-2 medium may suggest a regulatory role of the ammonium chloride-sensitive T cells in the AMLR.     

Cytofluorometric analyses of human T cell CD2/CD4 inter-molecular interactions

Incubation of human T lymphocytes with saturating concentrations of combinations of certain anti-CD2 and -CD4 mAb results in reciprocal down-regulation of the cell surface density expression of the respective CD molecules. Such reciprocal down-regulation occurs at 0 degrees C in the presence of sodium azide and appears selective for CD2 and CD4 molecules because mAb identifying various other CD T cell surface molecules (anti-Leu2a, -OK-CLL, -W6/32, -beta 2-microglobulin, -4B4) do not modulate CD2 or CD4 R density, and because anti-CD2 mAb (anti-OKT11 and -D66 clone-1) do not alter CD8 R density (anti-OKT8, -Leu2a) and vice versa. Down-regulation of CD2 by mAb specific to CD4 is epitope-specific but does not vary on the basis of the antibody isotype used. The anti-CD4 mAb, Leu3a, was the strongest CD2 down-regulator examined followed by OKT4F. mAb specific to other CD4 epitopes (B, C, D, and E) caused only slight down-regulation of CD2 expression whereas anti-OKT4 and -OKT4A mAb had no significant regulatory effect. Also, mAb specific to the 9.6 (anti-OKT11) and D66 (anti-D66 clone 1) epitopes of the CD2 molecule down-regulated CD4 density detectable with Leu3a, OKT4, and OKT4A anti-CD4 mAb. Down-regulation of CD2 by anti-CD4 mAb also occurred with the transformed T cell line, KE-37, which demonstrates that such effects can occur without mononuclear phagocytic accessory cells. From these data it can be concluded that important T cell immunoregulatory signals may be transmitted intramembranally between CD2 and CD4 glycoproteins.     

Lysis of fresh solid tumor targets in the presence of Con A is mediated primarily by Leu 7+ peripheral blood T lymphocytes: blocking by the anti-CD3 monoclonal antibody and comparison with recombinant interleukin 2-induced lysis by natural killer cells

We investigated the lysis of fresh human solid tumor cells by peripheral blood T lymphocytes in the presence of lectins and anti-CD3 monoclonal antibodies (mAb). Addition of certain lectins (Con A, PHA, or WGA) directly into the 4-hr 51Cr-release assay caused significant lysis of (P less than 0.001) noncultured solid tumor targets by enriched populations of granular lymphocytes (GL). Significant levels (P at least less than 0.001) of Con A- or PHA-dependent solid tumor lysis by GL-enriched lymphocytes were observed in 32 of 39 donors (82%) and 14 of 20 donors (70%), respectively. In contrast, the addition of other lectins (PNA, PWM, or LPS) or anti-CD3 mAb did not cause cytotoxicity. The levels of Con A-dependent lysis were comparable to those of interleukin 2 (IL-2)-induced lysis by Leu 11b+ natural killer (NK) cells. The presence of lectins at the effector phase, but not of recombinant IL-2 (rIL-2), was required for the lysis of solid tumor targets. Both Con A-dependent and rIL-2-induced lysis were totally inhibited by treatment of the effector cells with the lysosomotropic agent L-leucine methyl ester (LeuOMe). Effector cells responsible for Con A-dependent lysis of solid tumors expressed T3 (CD3), T8 (CD8), and Leu 7 antigens, but lacked T4 (CD4) and Leu 11 (CD16) antigens as determined by both negative and positive cell selection studies. Con A-dependent lysis was inhibited at the effector phase by anti-CD3 (OKT3 or anti-Leu 4) or anti-CD2 (OKT11) mAb. On the basis of their phenotype (Leu 7+ CD3+ CD8+ CD16-), we hypothesize that these effector cells may contain a population of cytotoxic T cells (CTL) generated in vivo against autologous modified cells that can lyse fresh solid tumor target cells under conditions where the recognition requirements for the CTL are bypassed by lectin approximation.     

Gamma-interferon-induced monocyte major histocompatibility complex class II antigen expression individuals with acquired immune deficiency syndrome

Twelve patients with the acquired immune deficiency syndrome (AIDS) and Kaposi's sarcoma were treated with recombinant human gamma-interferon (rIFN-gamma). A rapid, substantial increase in the fraction of HLA-DQ-positive monocytes was noted after treatment with rIFN-gamma. The rIFN-gamma-induced increase in monocyte HLA-DQ was seen throughout the course of treatment, with the percentage of HLA-DQ-positive monocytes dropping slightly following each week's treatment with rIFN-gamma and then rapidly increasing following the next course of treatment. Although the percentage of HLA-DR-positive monocytes was unchanged (HLA-DR was expressed on greater than 80% of monocytes prior to treatment), the density of HLA-DR on monocytes also increased following rIFN-gamma treatment. Following rIFN-gamma treatment, no changes were seen in CD3, CD4, CD8 T cell numbers, in T cell subset ratio (CD4/CD8), in Leu 7 or CD16 (Leu 11) cell number, in spontaneous Ig secretion, in PHA-induced in vitro proliferation, or in NK activity. These results indicate that exposure to rIFN-gamma in vivo led to the increased expression of class II antigens on monocytes in patients with AIDS.     

CD4 changes conformation upon ligand binding.

Aurintricarboxylic acid (ATA) has been shown to block the binding site for both HIV gp120 and mAb anti-Leu 3a on CD4. We have unexpectedly found that brief treatment with > or = 1 micrograms/ml ATA rapidly disengages another mAb, OKT4E, after it has been bound to CD4 on human PBL. OKT4E is specific for a discontinuous epitope overlapping the MHC class II-binding region in the N-terminal CD4 domain. Interestingly, among 10 other mAb tested, only anti-Leu 8, specific for a leukocyte homing receptor is also quickly released from the cells by ATA treatment. Disengagement of the OKT4E mAb is also seen on a CD4-positive cell line (HPB-ALL) and with recombinant soluble CD4 (sCD4) bound to immobilized OKT4E. In all of these cases, disengagement is prevented if OKT4E is cross-linked, or the Leu 3a site is blocked by the mAb, but not by gp120. Photobleaching fluorescence resonance energy transfer (pFRET) measurements suggest that OKT4E is released as an indirect consequence of ATA-evoked conformational changes of CD4. Similar changes were detected as a result of gp120 binding to PBL. These data raise the possibility of a novel type of immunomodulation: induced disengagement of a bound ligand from its Ag.     

Induction of suppressor cells to T- and B-cell proliferative responses and immunoglobulin production by monoclonal antibodies recognizing the CD3 T-cell differentiation antigen

Monoclonal antibodies (mAb's) recognizing the CD3 T-cell differentiation antigen induced the generation of suppressor cells. These cells inhibited (1) proliferative responses of human peripheral blood mononuclear cells (PBMC) to PHA and allogeneic cells in mixed leukocyte culture; (2) proliferative responses of purified E-rosette-negative cells to Staphylococcus aureus Cowans I; and (3) de novo immunoglobulin synthesis and secretion in the pokeweed mitogen (PWM)-induced differentiation system. Monoclonal antibodies recognizing other T-cell differentiation antigens (anti-Leu 2a, anti-Leu 3a, and anti-Leu 5) did not induce the generation of suppressor cells, even at very high antibody concentrations. Statistically significant differences were not observed in the ability of the OKT3 and anti-Leu 4 mAb's to induce suppressor cells. Monocytes were not required for the generation of anti-CD3-induced suppressor cells. F(ab')2 fragments of the OKT3 mAb's were equally effective when compared with intact antibody molecules in inducing suppressor cells, although they did not induce proliferative responses. Proliferation was not required for the induction of suppressor cells. Irradiation (2500 rad) of PBMC before incubation with the anti-CD3 mAb did not affect the generation of suppressor cells. Furthermore, anti-CD3-induced suppressor cells were radioresistant. Addition of recombinant IL-2 to the cultures of responding cells and suppressor cells did not reverse the suppression. In vitro treatment of anti-CD3-induced suppressor cells with either the OKT4 mAb plus complement or the OKT8 mAb plus complement partially decreased the suppression of proliferative responses of PBMC to PHA or allogeneic cells in mixed lymphocytes culture. However, treatment with both OKT4 and OKT8 mAb's plus complement or the OKT11 mAb plus complement completely abolished the suppression. These results suggest that the suppressor cells are of the T11+T4+T8- and T11+T4-T8+ phenotypes. In other experiments, T4+T8- and T8+T4- cells were isolated from PBMC treated for 48 hr with anti-CD3 mAbs. Both these two populations significantly inhibited proliferative responses of autologous PBMC to PHA and de novo immunoglobulin synthesis and secretion by mixtures of purified T4 and B cells from normal donors, in the PWM-induced differentiation system. These results demonstrate that anti-CD3-induced suppressor cells are of the T4 or T8 phenotype. Treatment of purified T4+T8- and T8+T4- cells with anti-CD3 mAb's resulted in the generation of suppressor cells, suggesting that the precursors of the anti-CD3-induced suppressor cells can belong to either of these two populations.(ABSTRACT TRUNCATED AT 400 WORDS)     

IFN-gamma and IFN-alpha induce the expression and synthesis of Leu 13 antigen by cultured human endothelial cells

Leu 13 is a 16 kDa human lymphocyte surface Ag and monoclonal anti-Leu 13 induces T cell aggregation and alters T cell proliferation. In previous studies using anti-Leu 13, Leu 13 Ag has been detected on endothelial cells (EC) of arteries, capillaries, and veins in human tissue sections. In contrast, in this study using the same antibody, Leu 13 Ag could not be detected on unstimulated cultured human umbilical vein endothelial cells (HUVEC) by indirect immunofluorescence, immunoperoxidase staining, or immunoisolation of radiolabeled Ag from labeled endothelial cells. However, when HUVEC were cultured with IFN-gamma, Leu 13 Ag was demonstrable using all three detection techniques. Leu 13 Ag was detectable after 24 h of incubation with IFN-gamma (30 U/ml) and was maximally expressed after 72 h. After removal of IFN-gamma, Leu 13 Ag progressively declined back to basal levels by 4 days. Induction of Leu 13 Ag expression by IFN-gamma was suppressed by cycloheximide. Although Leu 13 Ag expression was also induced by IFN-alpha, PMA, and IL-1 were inactive. When HUVEC were incubated with IFN-gamma, surface-labeled with 125I, and then solubilized, anti-Leu 13 immunoisolated a 16-kDa radioactive band that comigrated with Leu 13 immunoisolated from T cells. Similar results were obtained using HUVEC that were metabolically labeled with 35S-methionine. These results demonstrate that HUVEC stimulated with IFN-gamma and IFN-alpha express Leu 13 Ag on their surface and suggest that exposure of EC to IFN may induce EC to develop new functional properties at sites of inflammatory and immune reactions.     

Use of Leu3a/3b for the accurate determination of CD4 subsets for measurement of intracellular cytokines

BACKGROUND: Identification of human T-helper cell subsets is possible by measurement of intracellular cytokines after coincubation of lymphocytes with phorbol myristate acetate (PMA), calcium ionophore, and brefeldin A for up to 20 h. However, exposure to PMA leads to internalization of membrane CD4 and to loss of resolution of the CD4+ cells. Detection of CD3+CD8- cells or preselection of CD4+ cells prior to stimulation is more cumbersome than direct measurement of CD4+ cells. We report the use of the Leu3a/Leu3b multiclone for the accurate determination of CD4 cells after PMA stimulation. METHODS: Peripheral blood lymphocytes were isolated from healthy normal donors and the proportion of CD3+ / CD4+ T cells was determined by flow cytometry before and after incubation with PMA, calcium ionophore, and brefeldin A for 20 h using a variety of anti-CD4 monoclonal antibodies. RESULTS: The Leu3a/3b multiclone reagent was the only anti-CD4 monoclonal antibody capable of resolving more than 98% of the initial CD4+ events after incubation with PMA. CONCLUSIONS: The higher signal-to-noise ratio associated with Leu3a3b reagent, compared with other CD4-specific antibodies available, allows the direct and accurate identification of the CD4 subset even after PMA treatment of cells.     

Increased cytotoxicity and CD16 (Leu 11) expression in long-term, IL-2-activated human peripheral blood mononuclear cells

CD16 (Leu 11) positive cells are believed to be the effector cells for the so-called LAK phenomenon. Current evidence suggests that this cell population is comprised predominantly of IL-2-activated CD3 negative Leu 11+ NK cells and a minor proportion of Leu 11+ CD3+ MHC unrestricted type II cytotoxic T cells. The current study demonstrates a continuous increase in the frequency of Leu 11+ (and CD8+) cells and a decline of CD3 and CD4 positive cells during prolonged culture of human PMBL with high levels of rIL-2. Cytotoxicity also increases in this time period parallel with Leu 11 to a maximum of activity on the twelfth day of culture. This correlation suggests that the long-term activated killer cells generated in this period are Leu 11+, CD8+, CD3-, CD4- activated NK cells. With regard to tumor therapy, the long-term culture of PMBL in rIL-2 may be of advantage over short-term activation protocols. If the Leu 11+ cells are in fact the mediators of the therapeutic response, the long-term culture generates up to six times more effector cells. In addition, this method allows significant savings in the expense for leukophoresis, cell culture, and laboratory personnel. The efficacy of long-term, cultured rIL-2-activated Leu 11+ cells for tumor therapy is currently being investigated in clinical trails.     

Spontaneous human T-cell cytotoxicity against murine hybridomas expressing the OKT3 monoclonal antibody: comparison with natural killer cell activity

Human peripheral blood lymphocytes (PBL) exhibited spontaneous cytotoxicity against OKT3 monoclonal antibody (mAb)-expressing murine hybridoma cells (OKT3 hybridomas). In contrast, other murine hybridomas expressing OKT4, OKT8, anti-HLA DR, and anti-HLA A, B, and C mAb were not lysed. PBL showed much lower levels of cytotoxicity (3 folds) against OKT3 hybridomas as compared with NK activity against the K562 targets. Lymph node (LN) cells exhibited the inverse relationship of cytotoxicity levels. The addition of OKT3 mAb to the effector cells totally blocked both the binding and the lysis of OKT3 hybridoma targets, indicating that the CD3 antigen on the effector cells may be involved in recognition of the targets. The addition of concanavalin (Con A) also inhibited the cytotoxicity of OKT3 hybridomas. OKT4 mAb-expressing hybridomas became susceptible to lysis after chemical attachment of OKT3 mAb with CrCl3. The kinetics of lysis of OKT3 hybridomas resembled that of NK activity. Both cytotoxicities were detectable after 1 to 2 hr and reached plateau levels by 4 to 6 hr. Effector cells responsible for lysis of OKT3 hybridomas expressed T3, T8, and Leu 7 antigens, but lacked T4 and Leu 11b antigens, and were sensitive to the treatment with L-leucine methyl ester. These results indicate that T3+, T8+, Leu 7+ and T4-, and Leu 11- granular lymphocytes have a spontaneous cytotoxic activity against OKT3 hybridomas which is different from classic NK activity. These findings may provide a method for the assessment of T-cell cytotoxicity mediated presumably by in vivo generated cytotoxic T lymphocytes in blood and the other immune organs.     

Human cytotoxic lymphocytes. IV. Frequency and clonal specificity of CD8+CD16-(Leu2+Leu11-) and CD16+CD3-(Leu11+Leu4-) cytotoxic lymphocyte precursors activated by alloantigen or K562 stimulator cells

Cell sorter-purified CD8+CD16- (Leu2+Leu11-) cytotoxic T cell precursors and CD16+CD3-(Leu11+Leu4-) natural killer (NK) cells were cultured under limiting dilution (LD) conditions with allogeneic stimulator cells or with K562 tumor cells in the presence of exogenous interleukin 2. One out of 100-200 alloantigen-stimulated Leu2+ T cells clonally developed into an alloantigen-specific cytotoxic T cell, but only 1 out of 500-3400 of these cells lysed NK-susceptible K562 target cells. In contrast, 1 out of 2-35 alloantigen-stimulated Leu11+ precursor cells developed into an effector cell that lysed K562, but less than 1 out of 500 of these cells lysed allogeneic Con A blast targets. However, clonal activation of Leu11+ precursor cells under LD conditions did not require alloantigenic stimulator cells. Comparable high frequencies (f = 1/3 to 1/28) of anti-K562 cytotoxic lymphocyte precursors were thus measured when Leu11+ precursor cells were cultured on autologous or K562 feeder cells. As shown by a split culture approach, the vast majority of alloantigen-activated Leu2+ effector cells were highly specific for the stimulating alloantigen (i.e., they did not lyse K562), while the majority of Leu11+ microcultures lysed K562 tumor cells but neither autologous nor allogeneic Con A blast targets. On a quantitative basis, these data show that CD8+CD16- T cells and CD16+CD3-NK cells are two mutually exclusive lymphocyte populations which clonally develop into cytotoxic effector cells specific for alloantigen or K562 target cells, respectively.     

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.     

Polymorphonuclear neutrophils enhance anti-CD3-induced T cell activation: the role of polymorphonuclear FC-receptors.

We investigated the effect of polymorphonuclear neutrophils (PMN) on anti-CD3 mAb (OKT3 and anti-Leu4)-mediated T cell activation. In the absence of monocytes, purified E-rosette-positive cells (further referred to as "T cells") require either solid-phase bound anti-CD3 or the combination of both a high concentration of soluble anti-CD3 and exogenous recombinant interleukin 2 (rIL-2) to proliferate. PMN cannot sustain T cell proliferation with soluble anti-CD3, but they markedly boost proliferation in the presence of soluble anti-CD3 and rIL-2. When PMN were added to T cell cultures stimulated with anti-CD3, this resulted in IL-2 receptor (IL-2R) expression and CD3 modulation. The mechanism of enhancement of anti-CD3-induced IL-2-responsiveness by PMN was further analyzed. A cellular T cell-PMN interaction was found to play a critical role and this was mediated through PMN Fc receptors (FcR). PMN bear two types of low-affinity FcR (FcRII and FcRIII). FcRII is known to bind mIgG1 (e.g., anti-Leu4) and FcRIII binds mIgG2a (e.g., OKT3). FcR involvement was demonstrated by two observations. Anti-FcRII mAb IV.3 inhibited the PMN signal for T cell activation with anti-Leu4. PMN bearing the second variant of FcRII which is unable to bind mIgG1 failed to promote anti-Leu4/IL-2-mediated T cell proliferation. Thus, PMN potentiate T cell responsiveness to IL-2 in the presence of anti-CD3 mAb and this potentiation by PMN requires interaction of anti-CD3 with PMN-FcR.     

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.     

Human monocytes inhibit lymphokine-activated killer cell expansion in vitro

Depleting monocytes from human peripheral blood mononuclear cells (PBMC) enhances the in vitro activation of lymphokine-activated killer (LAK) cells. To determine if monocytes also altered LAK-cell expansion, we evaluated two methods of depleting monocytes from PBMC: nylon wool adherence (NWA) and phenylalanine methyl ester (PME) treatment. Both methods of depleting monocytes enhanced interleukin-2 (IL-2) driven, LAK-cell expansion; LAK expansion, however, was significantly greater after depletion with NWA than after PME. LAK cytotoxicity after NWA and PME depletion was equivalent. The degree of monocyte depletion, determined by evaluating morphology and the number of Leu-M3 (CD14) positive cells, and the proliferation of Leu 19 (CD56), OKT-3 (CD3), Leu2 (CD8), and Leu 3a (CD4) positive cells was also equivalent. Exposure of IL-2 activated cells to PME did not alter their cytotoxic activity. However, sequential treatment of PBMC with NWA, then PME, or with PME and then NWA, resulted in reduced expansion. This reduction in expansion was similar to PBMC treated with PME alone. Exposure of PME-depleted cells to nylon wool or to supernatants obtained from cells adherent to nylon wool further decreased LAK expansion relative to cells treated with NWA alone. We conclude that even at relatively low cell density, human monocytes markedly inhibit LAK-cell expansion in IL-2 driven PBMC cultures. Further, depletion of monocytes by NWA adherence is more effective than by treatment with PME, possibly due to subtle cellular damage induced by this latter treatment. These findings have implication for the in vitro and in vivo generation of LAK-cells by IL-2.     

A preliminary analysis of the balance between Th1 and Th2 cells after CD34+ cell-selected autologous PBSC transplantation

BACKGROUND: CD34+ cell-selected autologous PBSC transplantation (CD34+ APBSCT) is a procedure used for the treatment of patients with malignant disease that is intended to eliminate residual tumor cells from autologous grafts. However, frequent infectious complications after CD34+ APBSCT can occur. A delay of recovery of the absolute number of CD4+ T cells after transplantation was reported to be one disadvantageous factor. As data on T-cell function after CD34+ APBSCT are scanty, we analyzed changes in T-helper cell 1 (Th1) and T-helper cell 2 (Th2) after CD34+ APBSCT to evaluate immune reconstitution. METHODS: Twelve patients underwent APBSCT (CD34+APBSCT group, n=4, and unselected APBSCT, n=8). Peripheral blood (PB) samples were obtained at 2, 4, 8, 12 and 16 weeks after the transplantation. The dynamics of the Th1 and Th2 were analyzed at a single-cell level, using flow cytometry. RESULTS: In the CD34+ APBSCT group, not only the absolute count of CD4+ T cells but also the proportion of Th1 cells in CD4+ T cells and the ratio of Th1 to Th2 after transplantation were significantly decreased at 2 and 4 weeks after transplantation compared with findings in the unselected APBSCT group. DISCUSSION: We suggest that higher rates of infectious complications after CD34+ APBSCT may be due to the inability of residual T cells from the CD34+ cell selection to generate mature T cells that function adequately against infection. Although further study would be required, our preliminary data provide some information on the immune reconstitution after CD34+ APBSCT and differentiation of T lymphocytes into Th1 and Th2 in vivo.     

Association of human lymph node homing receptor (Leu 8) with the TCR/CD3 complex.

Cross-linking of the human homologue of the murine MEL-14 lymph node homing receptor (Selectin-1, LECAM-1, Leu 8) on both T and B cells results in modification of cell function. To investigate this phenomenon, we performed studies to determine if the Leu 8 molecule influences T cell activation via the TCR/CD3 complex. In initial studies, we treated T cells with immobilized anti-CD3 (OKT3 mAb) in the presence or absence of immobilized Leu 8 mAb. We found that although Leu 8 mAb alone had no effect on T cell proliferation, this antibody markedly augmented immobilized OKT3 mAb-induced proliferation. In further studies, we immunoprecipitated surface radioiodinated T cell lysates with OKT3 and Leu 8 mAb to determine if molecules in the TCR/CD3 complex associate with Leu 8 molecules. Although Leu 8 mAb immunoprecipitated only a single protein of approximately 80 kDa from T cell lysates treated with Nonidet P-40 under reducing condition, it coimmunoprecipitated additional proteins of 48, 42, 28, 24, and 22 kDa from T cell lysates treated with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate. These additional proteins were identified as the alpha-, beta-, gamma-, delta-, and epsilon-chains of the TCR/CD3 complex by one-dimensional and two-dimensional diagonal SDS-PAGE. Densitometric scanning showed that, on average, 18% of the TCR/CD3 complex associates with Leu 8. In a final study, we showed by immunoblotting analysis using anti-zeta peptide antibody that Leu 8 mAb coimmunoprecipitates the zeta-chain of CD3. These results indicate that the human lymph node homing receptor homologue (Leu 8) participates in the activation of T cells, probably via its association with the TCR/CD3 complex.