Simultaneous increased expression of E-rosette receptor (CD2, T11) and T cell growth factor receptor on human T lymphocytes during activation

The E-rosette receptor (CD2, T11) is a differentiation antigen expressed on immature and mature human T lymphocytes. Activation of T cells from human peripheral blood with phytohemagglutinin (PHA) or with monoclonal antibody to the CD3-Ti complex (anti-Leu-4) caused the expression of CD2 to increase 10- to 20-fold. Dual parameter correlated analyses with antibody to the T cell growth factor (TCGF) receptor (anti-Tac) and anti-CD2 antibody demonstrated that the increase in CD2 expression occurred at the same time and on the same cells that expressed the TCGF receptor after stimulation with PHA. The increased expression of CD2 and the initial expression of Tac were totally inhibited by cycloheximide, but were not affected by sufficient actinomycin-D to block the T cell proliferative response. The expression of CD2 was compared with the expression of CD4 and CD8, i.e., T cell differentiation antigens on cytotoxic/suppressor or helper T cells, respectively. Although virtually all of the small percentage of freshly isolated Tac+ peripheral blood cells belonged to the CD4+, CD8- subset, both CD4+ and CD8+ T cells were equivalently activated by PHA to express Tac. By 20-30 hr after activation, the expression of CD4 or CD8 was initially decreased 10-50%. Subsequently, the expression of CD4 and CD8 returned to the levels on resting T cells but did not increase further. Therefore, the increase in CD2 expression does not reflect a universal property of cell surface antigens on activated T lymphocytes.     

A distinct subset of human CD4+ cells with a limited alloreactive T cell receptor repertoire

We describe a subset of CD4+/CD3+ human T lymphocytes that demonstrated a remarkably limited TCR repertoire responding to alloantigen stimulation. These cells have been characterized previously by their granular morphology and expression of CD11b but not CD28. Whereas multiple CD28+/CD4+ alloproliferative cloned cell lines generated by culture at limiting dilution immediately after isolation from peripheral blood each had a unique TCR-beta gene rearrangement, 19 of 21 CD11b+/CD4+ clones showed identical TCR-beta, and gamma gene rearrangements. In conventional MLR, the CD11b+/CD4+ cells responded poorly after stimulation with some HLA-class II Ag, and staining with a TCR Id-specific antibody and DNA blot hybridization suggested that the responding CD11b+/CD4+ cells typically contained predominant clonal populations. Clones of CD11b+/CD4+ cells with different TCR gene rearrangements showed closely similar patterns of responses when stimulated by a panel of allogeneic PBMC, but the response pattern did not correspond to that of any known HLA-class II Ag. These findings indicate that CD11b+/CD4+ cells have a limited alloproliferative repertoire characterized by predominant recognition of a limited number of undefined determinants that appear to be expressed in association with multiple distinct HLA-class II Ag. Our results suggest that CD11b+/CD4+ cells are selected for clonal reactivity by processes distinct from those for CD28+/CD4+ cells.     

Reduced proliferation in T lymphocytes in aged humans is predominantly in the CD8+ subset, and is unrelated to defects in transmembrane signaling which are predominantly in the CD4+ subset

Peripheral blood lymphocytes (PBL) from elderly donors have a reduced proliferative response to phytohemagglutinin (PHA) and anti-CD3 monoclonal antibodies (mAb) compared to those from young donors. To examine whether this is due to intrinsic deficiencies in proliferative potential of T-cell subsets, we compared the growth of unsorted PBL vs sorted CD4+ or CD8+ CD11- cells after anti-CD3 mAb or PHA stimulation. Unsorted PBL of elderly donors (greater than 65 years) showed a significant decrease in proliferation compared to young donors (20-30 years) when stimulated with anti-CD3 mAb or PHA. Sorted CD4+ and CD8+ cells were grown in culture in the absence of accessory cells under optimized growth conditions (CD28 mAb, interleukin 2 and beta-mercaptoethanol present). CD4+ cells from elderly donors showed no reduced growth after anti-CD3 mAb stimulation and only slightly decreased growth after stimulation with PHA. CD8+ CD11- cells from elderly donors, however, showed a 20-30% reduction in the proportion of cells proliferating in response to the mitogens and up to 40% reduction in the rate of cell-cycle progression of the responding cells. We examined whether this reduced proliferation is related to decreased efficiency of signal transduction by comparing this to the mobilization of intracellular free calcium ([Ca2+]i) and calcium channel activity after stimulation with anti-CD3 mAb or PHA. [Ca2+]i was measured in CD4 and CD8 subsets of young and elderly donors using a flow cytometric assay with the dye indo-1. Compared to cells from young donors, CD4+ cells from elderly donors showed a [Ca2+]i response which was up to 26% lower after stimulation with CD3 and 10% lower after stimulation with PHA. This appeared to be related to decreased calcium channel activity in elderly donors, rather than mobilization of intracellular Ca2+ stores. CD8+ cells from elderly donors, however, had a slightly, but significantly, greater [Ca2+]i response to CD3 mAb and PHA than did cells from young donors. Since the age-dependent defect in proliferation is mainly in CD8+ cells, but the [Ca2+]i decline is predominantly in the CD4+ subset, these results suggest that the reduced proliferation of T cells from older donors is not related to decreased efficiency of transmembrane signal transduction.     

Production of lymphokines by circulating human T lymphocytes that express or lack receptors for interleukin 2

The capacity for circulating human T cells which have or lack receptors for interleukin 2 (IL 2) to produce IL 2, interleukin 3 (IL 3), and interferon-gamma (IFN-gamma) under the stimulus of phytohemagglutinin was studied. By using the monoclonal anti-Tac antibody which reacts against IL 2 receptors on human T cells, concanavalin A-treated T cells were separated into IL 2 receptor-positive (Tac+ T cells) and IL 2 receptor-negative (Tac- T cells) lymphocytes. The results show that Tac+ T cells secreted IL 2 and IFN-gamma but not IL 3. Tac- T cells produced IL 2 and IL 3 but not IFN-gamma. It is concluded that: 1) both T cells lacking and T cells having receptors for IL 2 produce IL 2, but only IL 2 receptor-negative T cells appear to secrete IL 3; and 2) virtually all of the T cells that produce IFN-gamma after PHA stimulation express receptors for IL 2.     

Valency of CD3 binding and internalization of the CD3 cell-surface complex control T cell responses to second signals: distinction between effects on protein kinase C, cytoplasmic free calcium, and proliferation

We have studied the relationship of valency of CD3 stimulation and modulation of the CD3 receptor complex with biochemical and proliferative responses of T cells. Anti-CD3 Fab, as well as F(ab')2 and whole antibody caused rapid modulation of the CD3 antigen, whereas anti-CD3 conjugated to Sepharose did not. In the absence of monocytes, T cells stimulated with anti-CD3 Fab, F(ab')2, or F(ab')2-Sepharose showed differences in their ability to respond to second signals given by PMA, IL 1, IL 2, or antibodies to Tp67 and Tp44. None of the anti-CD3 signals alone caused resting T cells to produce IL 2, and only the Sepharose-bound anti-CD3 F(ab')2 caused T cells to express high levels of functional IL 2 receptors. Anti-CD3 F(ab')2-Sepharose-stimulated T cells produced IL 2 and proliferated in response to each of the second signals. Because anti-CD3-Sepharose did not cause modulation of the CD3 antigen, the ability of the Sepharose-bound antibody to induce T cells to express IL 2 receptors and to respond to individual second signals may be related to lack of modulation rather than valency of binding. Anti-CD3 Fab-stimulated T cells responded to PMA but required combinations of other second signals. T cells stimulated with unmodified anti-CD3 antibody or F(ab')2 fragments responded to PMA but did not respond to any other second signals alone or in combination. Stimulations that resulted in modulation (i.e., anti-CD3 whole antibody, anti-CD3 F(ab')2, or anti-CD3 Fab fragments) caused an increase in cytoplasmic calcium levels in resting T cells but blocked proliferation of T cells in response to mitogenic lectins or CD2 stimulation. Anti-CD3 F(ab')2 on Sepharose, however, did not block T cell proliferation. Whole bivalent anti-CD3 antibody or F(ab')2 fragments, but not monovalent Fab fragments, caused a rapid translation of protein kinase C activity from cytosol to membrane in the Jurkat T cell line. Because all of these modulate the receptor, these data indicate that the functional difference between monovalent and bivalent binding to CD3 is related to antibody valency and not to antigenic modulation. The use of Fab anti-CD3 stimulation that requires combinations of second signals for proliferation allowed an analysis of the functional relationships between IL 1, anti-Tp67, and anti-Tp44.     

One-signal requirement for interferon-gamma production by human large granular lymphocytes

This laboratory has been investigating IFN-gamma gene expression by highly purified human large granular lymphocytes (LGL) and T cells. We report here that within 1 hr after interleukin 2 (IL 2) treatment of freshly isolated human LGL, IFN-gamma mRNA can be detected, with IFN-gamma protein in the culture medium within 4 to 6 hr of treatment. CD3- Leu-11+ LGL require only a single signal for IFN-gamma production because phytohemagglutinin (PHA), phorbol myristate acetate (PMA), IL 2, or ionomycin can each independently induce IFN-gamma production. In addition, PHA and ionomycin (but not IL 2) show significant synergy with PMA as a stimulus to LGL. In contrast, CD3+ T cells require two stimuli for high levels of IFN-gamma production, and not only are PMA plus ionomycin or PHA synergistic, but in addition, IL 2 and PHA demonstrate some synergy. Furthermore, we have found by fractionation of peripheral blood lymphocytes that IL 2-induced IFN-gamma production is associated with the LGL population and not T cells. These results indicate that with certain stimuli, LGL may be the predominant source of IFN-gamma from peripheral blood lymphocytes.     

IL-6/BSF-2 selectively stimulates the GO----S progression of CD8+ lymphocytes.

IL-6 preferentially promotes the DNA synthesis of human peripheral blood CD8+, rather than CD4+, lymphocytes in presence of PHA: this effect is observed in serum-free cultures of greater than 99% purified CD8+ lymphocytes. However, IL-6 is able to stimulate DNA synthesis of CD8+ lymphocytes triggered by a mitogenic anti-CD2 mAb, but not by anti-CD3 mAb: these results suggest that IL-6 selectively induces activation of CD8+ lymphocytes through the CD2 rather than the CD3 pathway. Limiting dilution analysis indicates that accessory cells are not required to mediate the action of IL-6 on CD8+ cells. Furthermore, this action is not blocked by addition of mAb neutralizing either IL-2 or IL2R, thus suggesting that IL-6 does not act via IL-2. CD8+ lymphocytes grown in the presence of PHA + IL-6 incorporate (3H)-thymidine to the same extent as those stimulated with PHA + IL-2, but do not increase in number until day 6 of culture. It is hence apparent that the stimulating activity of IL-6 on CD8+ lymphocytes is restricted to the GO----S phase progression, but does not lead to mitosis. IL-6 receptors are expressed on resting CD4+ and CD8+ lymphocytes: their expression is significantly enhanced on both activated CD4+ and CD8+ cells. Scatchard analysis of (125I)-IL-6 binding data showed the presence of high (Kd, 3 x 10(-10) M) and low (Kd, 6 x 10(-8) M) affinity IL6R on both lymphocyte populations. Similarly, mRNA encoding IL6R was detected in both CD4+ and CD8+ lymphocytes. Thus, our studies indicate that IL-6 directly and selectively stimulates the GO----S progression of CD8+ lymphocytes in the presence of mitogen and absence of IL-2: this phenomenon may be of interest for the elucidation of mechanisms activating cytotoxic T lymphocytes.     

Reactivity of lymphocytes to a progesterone receptor-specific monoclonal antibody

In this study we present evidence for reactivity of pregnancy lymphocytes, but not nonpregnancy lymphocytes, with the progesterone receptor-specific monoclonal antibody mPRI. Using an avidin-biotin peroxidase detection system, we found a nuclear staining in 14.6 +/- 3.7% (mean +/- SEM, N = 27) of pregnancy lymphocytes, while only 0.47 +/- 0.33% (mean +/- SEM, N = 15) of nonpregnancy lymphocytes reacted with the antibody. To characterize the receptor-bearing subset, CD8+ and CD4+ cells were depleted by complement-dependent lysis. Depletion of CD8+ cells was accompanied by 62 +/- 18% loss of progesterone receptor-bearing cells, while depletion of CD4+ cells resulted in a twofold increase in the number of positively staining lymphocytes. In nonpregnancy lymphocytes a 3-day PHA treatment, as well as allogeneic stimulation, resulted in a significant increase in the number of receptor-containing cells. These results suggest that pregnancy, but not nonpregnancy, lymphocytes contain progesterone binding structures, and that these are inducible by mitogenic or alloantigenic stimuli.     

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.     

Cytotoxic T lymphocyte triggering via CD16 is regulated by CD3 and CD8 antigens. Studies with T cell receptor (TCR)-alpha beta+/CD3+16+ and TCR-gamma delta+/CD3+16+ granular lymphocytes

The role of CD3 and CD8 Ag in CD16-mediated CTL triggering was studied in TCR-alpha beta+ and TCR-gamma delta+ granular lymphocytes (GL). In TCR-alpha beta+/CD3+4-8+16+ GL obtained from patients with GL-proliferative disorders, antibody-dependent cellular cytotoxicity was inhibited by anti-CD3 and anti-CD8 mAb. Anti-CD3 mAb also inhibited antibody-dependent cellular cytotoxicity activity of TCR-gamma delta+/CD3+4-8-16+ GL from a patient and that of TCR-gamma delta+/CD3+4-8+/-16+ T cell clones established from patients with proliferating TCR-gamma delta+ GL. In TCR-gamma delta+ T cell clones, cytotoxicity against Fc gamma R+ targets was induced by stimulation of CD16 Ag with anti-CD16 mAb, and such cytotoxicity was also inhibited by anti-CD3 mAb. These results indicate that CD3 and CD8 molecules play a regulatory role in CD16-mediated CTL triggering.     

Lysis of tumor cells by CD3+4-8-16+ T cell receptor alpha beta- clones, regulated via CD3 and CD16 activation sites, recombinant interleukin 2, and interferon beta 1

A small subpopulation (about 2%) of normal CD3+ human T lymphocytes lacks both CD4 and CD8 antigens. We have cloned these cells from peripheral blood lymphocytes (PBL) obtained from healthy individuals and from a patient with severe combined immunodeficiency. Six out of seven CD3+4-8-clones exert strong cytolytic activity against a variety of so-called NK-susceptible and -nonsusceptible tumor target cells. Their target cell specificity spectrum can virtually be as wide as that of CD3-NK cell-derived clones, with strong lytic capacity. Some of these clones also exert antibody-dependent cellular cytotoxicity (ADCC), a characteristic of NK cell-derived clones but not of CD3+4+ or CD8+ mature T cell-derived clones. Such CD3+ T cell clones do not express the CD16 (IgG Fc receptor) antigen, but as we demonstrate here, the CD16 antigen can be identified on CD3+4-8-clones. Both ADCC activity and CD16 antigen expression are lower in CD3+4-8- than in CD3- NK cell clones. Lytic activity of mature CD3+4+ or CD8+ and CD3- NK cell clones can be augmented, respectively, by anti-CD3 or anti-CD16 monoclonal antibodies (MAb), but that of CD3+4-8- clones are augmented by both MAb. Lytic activity of CD3+4+ or CD8+ clones is considerably enhanced after 3 hr of incubation with recombinant IL 2, as found for CD3- NK cells. Enhancement of lytic activity of allospecific CD3+4+ or CD8+ clones requires 18 hr of incubation. Thus, CD3+4-8-16+ cells share several features with CD3- NK cells. However, they express the CD3 antigen, which is characteristic for CD4+ or CD8+ mature T cells. Our results also indicate that although CD3+4-8- clones react with five preparations of anti-CD3 MAb tested, these clones do not express a classical CD3+/Ti alpha, beta antigen receptor complex. This is suggested by the finding that the CD3+4-8- clones do virtually not express the common epitope of the T cell receptor alpha, beta-chains as identified by the WT31 MAb. These CD3+4-8- lymphocytes may represent functionally mature lymphocytes of a distinct T cell subpopulation having a particular immune function.     

Comparison of T cell receptor gene rearrangements in patients with large granular T cell leukemia and Felty's syndrome

Felty's syndrome (FS) refers to the occurrence of rheumatoid arthritis, splenomegaly, and neutropenia. A subset of these patients has recently been described with a chronic T cell leukemia of large granular lymphocytes (LGCL). To examine the spectrum of lymphocyte abnormalities in FS and LGCL, we examined phenotypic and genotypic properties of lymphocytes from eight FS patients. In two of these FS patients, we observed an elevated proportion of T cells with an unusual phenotype (CD3+/Leu-7+/Leu-8-/CR3+) (46 +/- 5% of mononuclear cells). The FS lymphocytes had large granular morphology on Wright-Giemsa stain and were active in antibody-dependent cellular cytotoxic activity. This phenotype, morphology, and activity was similar to LGCL patients except that the latter T cells additionally expressed the Fc-IgG receptor recognized by monoclonal antibody Leu-11 (CD 15). In the remaining six FS patients, the proportion of CD3+/Leu-7+/CR 3+ T cells was only 10 +/- 8%, which was not significantly different from age-matched normal subjects (6.6 +/- 2.2%). To determine the clonality of T lymphocytes in FS and LGCL, we examined DNA for rearrangements of the T cell antigen receptor beta-chain (Ti beta) and gamma-chain (Ti gamma) genes by using Southern blotting techniques. We found a clonal rearrangement of the Ti beta 1 and Ti gamma genes in both LGCL patients. In contrast, no clonal rearrangements of Ti beta or Ti gamma genes were detected in lymphocytes from the FS patients. These results indicate that FS patients are heterogeneous in their phenotype and that one subset exhibits polyclonal expansion of an unusual lymphocyte subset.     

Human peripheral blood T helper cell-induced B cell activation results in B cell surface expression of the CD23 (BLAST-2) antigen

We have developed an in vitro system to assess the early stages of B cell activation induced by peripheral blood T helper cells. Peripheral blood mononuclear cells are cultured for 16 hr with anti-CD3 monoclonal antibody (mAb), T lymphocytes are then removed by sheep red blood cell rosette depletion, and expression of the B cell surface activation antigen CD23 (BLAST-2) is assessed by indirect immunofluorescence. Anti-CD3 mAb, but not a control anti-CD5 mAb, stimulates the expression of CD23 on 20-50% of peripheral blood B cells cultured with autologous T cells. T cell subset depletion studies show that the CD4+ T cell subset is responsible for anti-CD3-mediated induction of CD23 on autologous B cells. Anti-CD3-induced, T helper cell-dependent CD23 expression is not MHC-restricted, as allogeneic combinations of T and non-T cells, cultured in the presence of anti-CD3 antibody, also result in the expression of B cell CD23. Individuals whose monocyte Fc receptors bind murine IgG1 mAb poorly fail to trigger T cell proliferation in response to murine IgG1 anti-CD3 mAb and also fail to express B cell CD23 following culture of PBMC with IgG1 anti-CD3 mAb, while the usual expression of CD23 is seen after culture with IgG2a anti-CD3 mAb. The mechanism of anti-CD3-induced B cell activation was addressed in experiments using a two-chamber culture system. While little IL-4 activity was detected in anti-CD3-stimulated culture supernatants, optimal induction of CD23 was observed when T and B cells were cultured together in a single chamber. This suggests that under physiologic conditions, in which quantities of lymphokine may be limiting, close physical contact between the anti-CD3-activated Th cell and B cell may be required for CD23 expression. The anti-CD3-induced BLAST-2 assay will facilitate the analysis of Th cell-mediated B cell activation in any individual and should permit us to separately evaluate the roles of Th cells and B cells in the impaired immunoregulation characteristic of autoimmune disorders.     

Involvement of beta 2-integrins in the migration of human natural killer cells.

Human large granular lymphocytes with the NK cell phenotype (CD16+ or CD56+CD3-) were greatly enriched among the cells which migrated spontaneously through untreated or albumin-coated, 3-microns pore size polycarbonate filters for 1 to 8 h. Three days of rIL-2 treatment (300 IU/ml) and 3 to 5 wk of rIL-2 treatment (100 IU/ml) generated a 2.7 +/- 0.9-fold and 5.6 +/- 0.8-fold increase in cell migration, respectively. The adhesion and subsequent migration of freshly isolated NK cells was mainly mediated by CD11b/CD18, because migration could be inhibited by 80 +/- 8% anti-CD11b (Mac-1) antibodies but not with antibodies against CD11a (LFA-1) or CD11c (p150,95), the other alpha-chains of the beta 2-integrins. After rIL-2 activation, however, CD11a/CD18 was the major receptor utilized in migration, inasmuch as anti-CD11a antibody caused a 69 +/- 8% reduction in the number of migrated cells. Anti-CD11b antibody decreased migration by 43 +/- 12%, and together these antibodies inhibited migration by 82 +/- 7%. Anti-CD11a alone did not have any effect on adhesion, but CD11a/CD18 cooperated in the adhesion because anti-CD11b decreased adhesion by 40 +/- 11% and together these antibodies inhibited adhesion by 74 +/- 6%. The ability of large granular lymphocytes to rapidly utilize beta 2-integrins and unidentified ubiquitous ligands for binding and migration may be significant for their capacity to function in the first line of immune defense under highly variable conditions.     

The lymphocyte-specific tyrosine protein kinase p56lck is endocytosed in Jurkat cells stimulated via CD2.

Incubation of the human T cells, Jurkat, with two sets of activating anti-CD2 mAb (T11(2) + T11(3), D66 + T11(1)) induced delocalization of p56lck and CD2 receptors from the plasma membrane and increased the tyrosine kinase activity of p56lck. The anti-CD2 mAb combination (T11(2) + T11(3)) that produced the most rapid increase in p56lck kinase activity also induced the most rapid delocalization of the kinase. In stimulated cells, both p56lck and CD2 receptors are detected in cytoplasmic vesicles. The internalization of p56lck in endocytic vesicles was established by confocal microscopy. By double staining it was shown that only part of the p56lck colocalized with the internalized CD2 receptor suggesting distinct sorting processes. Internalization of p56lck appeared to be specific of CD2 stimulation as: 1) in Jurkat cells triggered with an anti-CD3 mAb, p56lck was not internalized whereas CD3 receptors were completely endocytosed; 2) when cells were stimulated via CD4, the kinase and CD4 receptors remained associated with the plasma membrane. In addition, internalization of p56lck upon stimulation of CD2 receptors was not modified in CD2+/CD3-Jurkat cells indicating that CD3 is not involved in this process. The identification of different subcellular localizations of p56lck in resting and stimulated T cells should represent an important step in the definition of its functional activity.