Two-color flow cytometry and functional analysis of lymphocytes cultured from human renal allografts: identification of a Leu-2+3+ subpopulation

The phenotype of T lymphocyte subsets present in renal biopsies showing acute cellular allograft rejection in six patients on cyclosporine have been characterized in situ by immunoperoxidase staining, and after expansion in vitro in interleukin 2 (IL-2) by two-color flow cytometry, sorting, and functional analysis. After 8 to 42 days in organ culture, both Leu-3+ (CD4) and Leu-2+ (CD8) subsets were found in each culture, in a ratio that varied from 0.2 to 5.0, which was not significantly different than the results of in situ immunoperoxidase staining of the uncultured biopsy. The cultured cells were almost all Leu-4+ (CD3) T cells (89% +/- 4), which expressed the activation markers DR (82% +/- 6) and the IL 2 (CD25) receptor (15% +/- 4). The Leu-3+ cells were largely Leu-8- (90% +/- 6), whereas a minority of the Leu-2+ cells were Leu-15+ (CD11) (26% +/- 4). Only a small fraction of the Leu-2+ cells stained for Leu-7 (8% +/- 6). Functional analysis of FACS-purified Leu-2-3+ and Leu-2+3- populations indicated that both subsets proliferated in response to graft donor antigens in a mixed lymphocyte reaction (MLR) and produced IL 2. Only the Leu-2+3- population demonstrated donor-specific cytotoxic activity. A minor subpopulation in each culture were both Leu-3+ and Leu-2+ (2.0%). Leu-2+3+ cells from one biopsy were purified to homogeneity (99.8%), and were found to express the T cell antigen receptor complex Ti/CD3 (WT-31+, Leu-4+), but not the common thymocyte antigen CD1 (OKT6). The Leu-2+3+ cells neither responded in the MLR, nor showed any cytotoxic capacity. The Leu-2+3+ cells were capable of IL 2 but not interferon-gamma production. None of the purified cultures demonstrated NK activity. A subset of the purified Leu-2+3+ cells lost Leu-2+ during 1 to 3 wk in culture, and became Leu-2-3+. These studies provide evidence that the cells that infiltrate renal allografts during rejection include alloproliferative, lymphokine-producing cells of both Leu-2+ and Leu-3+ subsets. The Leu-2+3- cells are also highly cytotoxic against donor lymphocytes, indicating the presence of helper independent cytotoxic T cells. A minor population of Leu-2+3+ T cells that do not express donor specific function was also identified.     

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

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

Studies on human blood lymphocytes with iC3b (type 3) complement receptors. I. Granular, Fc-IgG receptor positive and negative subsets in healthy subjects and patients with systemic lupus erythematosus

By using the OKM1 monoclonal antibody and the fluorescence-activated cell sorter to identify lymphocytes bearing iC3b (type 3) complement receptors, two principal populations of OKM1+ lymphocytes have been identified in human peripheral blood. One subset exhibited azurophilic granules and Fc receptors for IgG stained by Leu-11. The other population did not display FcR, but was enriched in cells reacting with OKT3 and OKT8 (low intensity). In healthy subjects, approximately 60% of CR3+ lymphocytes were granular FcR-bearing cells and only 18% co-expressed OKT3 determinants. In patients with systemic lupus erythematosus (SLE), CR3+ lymphocytes were predominantly FcR negative cells and 71% lacked granules. Only 33% reacted with Leu-11, but 50% co-expressed OKT3, 44% reacted with OKT8+, and 15% were OKT4+. We tested the hypothesis that agranular OKT3+ Leu-11- lymphocytes, such as those found in SLE patients, contained the precursors of natural killer (NK) cells. Leu-11+ cells were removed from normal lymphocytes by complement lysis, and the remaining cells were treated with recombinant IFN-alpha, IFN-gamma, or IL 2. These procedures were ineffective in generating typical NK effector cells. Our studies do not support the hypothesis that CR3+ Leu-11- lymphocytes are the precursors of granular Leu-11+ NK cells.     

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.     

Predictable alphabeta T-cell receptor selection toward an HLA-B*3501-restricted human cytomegalovirus epitope

Human cytomegalovirus (HCMV) elicits a very large burden on the immune system, with approximately one in ten T cells being reserved solely to manage this infection. However, information on the clonotypic composition of these vast T-cell populations is limited. In this study, we sequenced 116 T-cell receptor (TcR) alpha/beta-chains specific for the highly immunogenic HLA-B*3501-resticted epitope IPSINVHHY from the pp65 antigen. Interestingly, T cells recovered from all donors bore an identical or near-identical TRBV28/TRBJ1-4/TRAV17/TRAJ33 TcR. The ability to predict the responding alphabeta TcR repertoire before viral infection should prove a powerful tool for basic and clinical immunology.     

The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes

We examined the antigenic and functional characteristics of human peripheral blood lymphocytes that differentially express the CD16 (Leu-11) and Leu-19 (NKH-1) antigens. Leu-19 is a approximately 220,000 daltons protein expressed on approximately 15% of freshly isolated peripheral blood lymphocytes. Within the Leu-19+ subset, three distinct populations were identified: CD3-,CD16+,Leu-19+ cells; CD3+,CD16-,Leu-19+ cells; and CD3-,CD16-,Leu-19bright+ cells. Both the CD3+,CD16-,Leu-19+ and CD3-,CD16+,Leu-19+ populations mediated non-major histocompatibility complex (MHC)-restricted cytotoxicity against the NK-sensitive tumor cell K562 and were large granular lymphocytes. CD3-,CD16+,Leu-19+ NK cells were the most abundant (comprising approximately 10% of peripheral blood lymphocytes) and the most efficient cytotoxic effectors. The finding that CD3+,Leu 19+ lymphocytes mediated cytotoxicity against K562 unequivocally demonstrates that a unique subset of non-MHC-restricted cytotoxic CD3+ T lymphocytes are present in the peripheral blood of unprimed, normal individuals. However, CD3+,CD16-,Leu-19+ cells comprised less than 5% of peripheral blood lymphocytes, and the cytotoxic activity of this subset was significantly less than CD3-,CD16+,Leu-19+ NK cells. Most CD3+,Leu-19+ T cells co-expressed the CD2, CD8, and CD5 differentiation antigens. The antigenic and functional phenotype of peripheral blood CD3+,Leu-19+ cytotoxic T lymphocytes corresponds to the interleukin 2-dependent CD3+ cell lines that mediate non-MHC-restricted cytotoxicity against NK-sensitive tumor cell targets. A small population of Leu-19bright+ lymphocytes lacking both CD3 and CD16 was also observed. This population (comprising less than 2% of peripheral blood lymphocytes) contained both large agranular lymphocytes and large granular lymphocytes. CD3-,CD16-,Leu-19bright+ lymphocytes also mediate non-MHC-restricted cytotoxicity. The relationship of these CD3-CD16-,Leu-19bright+ lymphocytes to CD3+ T cells or CD16+ NK cells is unknown.     

Establishment and characterization of a clonal human T-cell line, MKB-1 derived from a patient with acute myeloblastic leukemia

A human T-cell line, designated as MKB-1, was established by cloning procedures in a suspension culture from a peripheral blood of a 17-year-old female patient with acute myeloblastic leukemia. The immunological marker profile of MKB-1 indicated that unlike a myeloid phenotype of the original leukemic cells, the cells were positive for CD3 (both cell surface and cytoplasm), T cell receptor (TcR) alpha/beta heterodimer, CD4, CD5, CD7, CD10, CD57 (Leu7), SN-1 and the cytoplasmic TcR beta chain. These findings indicate the T cell nature of the established cells. Terminal deoxynucleotidyl transferase (TdT) was also detected in 60%. We did not detect markers of human myeloid and B cell associated antigens, HLA-class II or immunoglobulin chains. Cytogenetic study revealed that the MKB-1 cells had a female hypo-tetraploid karyotype with chromosomal abnormalities including a translocation between chromosomes 10 and 14. The breakpoint of chromosome 14 of this translocation, 14q11.2, is known to be the location of TcR alpha and delta genes; t(10; 14) (q26; q11.2) is a variant type of a T cell neoplasm-associated translocation, t (10; 14) (q24; q11.2). The MKB-1 cell line is unusual in that its T cell characteristics are phenotypically and cytogenetically distinct from the original myeloid leukemia cells.     

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.     

Characterization of recirculating lymphocytes in rheumatoid arthritis patients: selective deficiency of natural killer cells in thoracic duct lymph

Five patients with rheumatoid arthritis (RA), who were treated by lymphocyte depletion by using thoracic duct drainage (TDD), provided an opportunity to characterize the phenotype and function of their recirculating lymphocytes. We found that: a) thoracic duct lymphocytes (TDL) were similar in their proportion of T cells (83% +/- 6 OKT3+), OKT4+ subset (65% +/- 8), and OKT8+ subset (22% +/- 6) to peripheral blood lymphocytes (PBL): b) fewer natural killer-like cells were present in TDL (5% +/- 4 Leu-7+; 2% +/- 2 Leu-11+: 8% +/- 2 OKM -1+) than in PBL (20% +/- 10 Leu-7+: 11% +/- 6 Leu-11+; 18% +/- 5 OKM -1) (p less than 0.01); c) TDL differed from synovial fluid lymphocytes ( SFL ) and synovial membrane lymphocytes ( SML ) in that TDL lacked a high percentage of activated lymphocytes (T cells bearing Ia antigen, OKT10 , and transferrin receptor): d) immature T cells (expressing either OKT6 antigen or reactive with peanut agglutinin) were not found in TDL even late in the course of TDD: and e) in vitro functional studies demonstrated that TDL were similar to PBL in their ability to synthesize immunoglobulin after mitogen stimulation and to generate cytotoxic T lymphocytes capable of lysing autologous EBV-transformed B cells. However, natural killer activity, as measured by lysis of K562 cells was significantly lower in TDL than PBL (p less than 0.05). These results demonstrate that natural killer cells defined by phenotype and function are excluded from thoracic duct lymph and thus have a circulation pattern different from most T cells.     

Lateral diffusion of CD4 on the surface of a human neoplastic T-cell line probed with a fluorescent derivative of the envelope glycoprotein (gp120) of human immunodeficiency virus type 1 (HIV-1)

The envelope glycoprotein (gp120) of HIV-1 was labeled with fluorescein by using 6-[4,6-dichlorotriazinyl]aminofluorescein. The labeled glycoprotein was found to bind to CD4-positive CEM cells. Monoclonal antibody OKT4a but not OKT4 blocked this binding. Similar specific binding of fluorescein-labeled gp120 with CD4 was observed in a solid-phase ELISA where sCD4 was attached to a polystyrene plate. The syncytium formation induced by HIV-1-infected cells on CEM cells was significantly inhibited in the presence of fluorescein-labeled gp120. Fluorescence photobleaching recovery measurements showed that the diffusion coefficient (D) of CD4 molecules complexed with fluorescein-labeled gp120 was approximately 5 x 10(-10) cm2sec-1, with nearly 61% of the receptor molecules being mobile. Binding of anti-gp120 monoclonal antibody to the CD4-gp120 complex reduced the mobile fraction significantly. Diffusion of CD4 labeled with OKT4 IgG was markedly inhibited with reductions in both D and the mobile fraction, but such inhibition was not observed with OKT4 Fab. It appears that crosslinking of multiple molecules of CD4 by OKT4 antibody is required to reduce CD4 mobility. This suggests that the receptor might be present on the membrane plane as molecular clusters containing at least two molecules of CD4.     

Is a natural ligand of the T lymphocyte CD2 molecule a sulfated carbohydrate?

Recent studies have implicated sulfated polysaccharide (SP) recognition in a range of cell adhesion systems. Inasmuch as the CD2 (E rosette receptor, T11, LFA-2) molecule of human T lymphocytes is a cell surface glycoprotein involved in the adhesion of T cells to various target cells the possibility that CD2 binds SP was investigated. It was found that E rosetting of human T lymphocytes, a phenomenon involving CD2, was readily inhibited by the SP dextran sulfate (DxS) and, to a lesser extent, by the sulfated polymer polyvinyl sulfate whereas 11 other SP had no effect on E rosetting, this effect occurring at the T cell level. mAb binding studies revealed that DxS and polyvinyl sulfate, but none of the other SP tested, inhibited the binding to T cells of the anti-CD2 mAb OKT11 and anti-T112 but augmented expression of the T113 epitope of the CD2 molecule. In contrast, DxS had little or no effect on the binding of anti-CD3, -CD4, -CD8, -Pgp-1 and WT31 (TCR alpha/beta) mAb. Direct evidence that CD2 binds DxS was demonstrated by the ability of DxS-coupled fibers to totally deplete the CD2 Ag from lysates of radiolabeled human T lymphocytes and by the quantitative recovery of the CD2 Ag in fiber eluates. Control fibers coupled with other SP bound little or no CD2. Collectively, the data indicate that the CD2 molecule specifically binds DxS and suggest that a potential target cell ligand for CD2 is a sulfated carbohydrate structure.     

A DC-specific cytolytic T lymphocyte line is OKT8+1

A human cytotoxic T lymphocyte (CTL) line, A9, was generated by limiting dilution and was selected because of its apparent DC specificity. A9 is 100% OKT3+, 90% OKT4+, and 10% OKT8+, but by negative selection the CTL present are entirely OKT8+. These OKT8+ CTL are totally inhibitable by Genox 3.53, an anti-DC1 monoclonal antibody (mAb), and Leu-10, an anti-DC subgroup mAb, but are not inhibitable by a panel of anti-HLA-DR mAb. These CTL are also inhibitable by anti-OKT3 and anti-LFA-2 but not by OKT4 or OKT8 mAb. These findings extend previous studies that showed that OKT8+ CTL recognize HLA-A,B antigens, whereas OKT4+ CTL recognize HLA-DR and SB antigens. It is possible that an as yet undefined T cell surface molecule is involved in DC recognition.     

Immature T lymphocytes in human cord blood identified by monoclonal antibodies: a model for the study of the differentiation pathway of T cells in humans

The reactivity of human cord blood lymphocytes was assessed against a panel of monoclonal antibodies (MoAb). The mean proportion of OKT3+ cells (pan-T) was significantly lower in cord blood (52 +/- 13.8%; mean +/- SD) compared with that of adult blood (75 +/- 8.9%) and paralleled well with the E-rosette-forming capacity (50 +/- 16.3%). Both the proportions of OKT4+ cells (helper/inducer phenotype) and of OKT8+ cells (suppressor/cytotoxic phenotype) were significantly reduced in cord blood (43 +/- 11.8% vs 50.3 +/- 7.4% and 20 +/- 10.3% vs 25.6 +/- 6.0%, respectively), while the overall OKT4/OKT8 ratio was increased compared with adult blood (2.87 +/- 1.83 vs 2.04 +/- 0.61). Unlike adult blood, in 30 of the 35 samples of cord blood an overlap was observed between the total proportion of OKT4+ and OKT8+ cells (65 +/- 15.2%) and that of OKT3+ cells (52 +/- 14.3%). Although small numbers of cells coexpressing both antigens were occasionally found, double-staining analysis showed that the overlap in cord blood was mostly due to an expanded proportion of OKT3 (Leu-4)-/OKT8 (Leu-2)+ cells. Relevant proportions of OKT6+ (common thymocyte antigen) and OKT10+ (thymocytes, activated T cells, precursor cells) cells were found in cord blood as opposed to adult blood (10.8 +/- 8.6% vs 0.6 +/- 0.6% and 67 +/- 18.0% vs 8 +/- 2.1%, respectively), while terminal deoxynucleotidyl transferase-positive cells were observed only in two samples of cord blood. A small proportion of T cells (E-rosette+) reacted with the MoAb OKIa1 (HLA-DR). Finally, the proportion of cord blood cells recognized by the MoAb Leu-7 (HNK-1 clone) was almost negligible compared with adult blood (2.8 +/- 2.4% vs 15 +/- 7.5%). These data confirm the immaturity and heterogeneity of cord blood lymphocytes and demonstrate the presence at birth of circulating lymphocytes which express a surface phenotype reminiscent of that found in the late stages of intrathymic differentiation and in some human T-cell leukemias. Human cord blood may thus represent a suitable model for the study of the differentiation pathway of normal and pathological T-cells in humans.     

Accessory cell-T cell interactions involved in anti-CD3-induced T4 and T8 cell proliferation: analysis with monoclonal antibodies

The effect of monoclonal antibodies (Mab) directed at T cell and accessory cell (AC) surface molecules on OKT3-induced T4 and T8 cell proliferation was examined. Mab directed at nonpolymorphic class I (W6/32, MB40.5) and class II (L243) major histocompatibility complex (MHC)-encoded gene products, an epitope common to LFA-1, CR3, and the p150, 95 molecule (60.3), and a heterodimer present on monocytes (M phi) and activated T cells (4F2) inhibited M phi-supported OKT3-induced proliferation of both T4 and T8 cells. Moreover, an Mab directed at the CD4 molecule (66.1) inhibited OKT3-induced T4 but not T8 cell proliferation, whereas an Mab directed at the CD8 molecule (OKT8) inhibited T8 but not T4 cell responses. With the exception of 66.1, each inhibited OKT3-induced T cell proliferation when added as late as 15 hr after the initiation of culture. Inhibition could not be explained by competition for Fc receptors on the AC. A variety of other Mab including OKT11 and those directed at other HLA-DR and DQ determinants were not inhibitory. The inhibitory Mab were found to diminish T4 cell IL 2 production and IL 2 receptor expression. Consequently, IL 2 reversed some but not all of the Mab-mediated inhibition of T cell proliferation. In contrast to the effects noted with M phi-supported responses, 60.3 and 66.1 but neither L243 nor 4F2 inhibited OKT3-induced T4 cell proliferation supported by Ia- or IFN-gamma-treated Ia+ endothelial cells. None of the Mab tested inhibited T cell proliferation induced by the AC-independent stimuli OKT3 and phorbol myristate acetate (PMA) or calcium ionophore and PMA in the presence or absence of added AC. The data therefore suggest that the Mab inhibit OKT3-induced activation of T4 and T8 cells by preventing necessary interactions between AC and T cell surface proteins. Moreover, the results suggest that different arrays of interaction molecules are involved in OKT3-induced T cell proliferation depending on the nature of the AC and the responding T cell subset.     

Regulation by anti-CD2 monoclonal antibody of the activation of a human T cell clone induced by anti-CD3 or anti-T cell receptor antibodies

In this study the effect of anti-cluster designation (CD) 2 monoclonal antibodies (mAb) on the activation of a cloned human T cell line, HY837, after triggering the CD3/T cell receptor (TcR) complex by anti-CD3 or anti-TcR mAb is described. HY837, which reacts with a series of mAb directed at different epitopes on the TcR, could be induced to proliferation and interleukin 2 (IL-2) production by soluble mAb directed at the CD3/TcR complex in the absence of accessory cells. mAb directed at the CD2 epitope T11-1 were shown to block the IL-2 production by HY837, as well as the expression of the IL-2 receptor, induced by anti-CD3 mAb, resulting in the inhibition of the proliferative response. The effect of anti-CD2 mAb on the proliferative response of HY837, induced by anti-CD3 mAb, was not due to a competition for Fc binding sites. In contrast, the proliferative responses and IL-2 production of HY837, induced by mAb directed at the TcR, were shown to be enhanced by the action of the anti-CD2 mAb. These results indicate that effects mediated by anti-CD3/TcR mAb cannot always be extrapolated to antigen-mediated effects and show that anti-CD2 mAb may regulate the T cell response, induced by mAb directed at the CD3/TcR complex, depending on which part of this complex is triggered during activation.     

Human peripheral blood leukocyte engraftment into SCID mice: critical role of CD4(+) T cells

We examined the influence of donor T lymphocytes on human peripheral blood leukocytes (PBL) engraftment into severe combined immune deficient (SCID) mice. Mice were injected with unfractionated or subset-depleted human PBL, and treated at various times with OKT3, a cytotoxic monoclonal antibody against human CD3(+) T lymphocytes. PBL engraftment, high levels of human Ig, and high incidence of lymphoproliferative disease (lpd) were found in mice transplanted with unfractionated PBL and CD8- or CD14-depleted PBL, and in mice treated with OKT3 at distance from PBL transfer. Animals xenografted with CD3- or CD4-depleted PBL, or treated at transplantation time with OKT3, had very low levels of human Ig and did not develop lpd. PBL engraftment was minimal or absent in these animals, as determined by immunohistochemistry, dot-blot, and RT-PCR analyses. These results demonstrate that the presence of donor CD4(+) T lymphocytes at transplantation time is necessary for observing human PBL engraftment into SCID mice, an essential condition for human Ig production and lpd development.     

Mononuclear cell subpopulations and infiltrating lymphocytes in erythema dyschromicum perstans and vitiligo

Erythema dyschromicum perstans (EDP) and vitiligo are two cutaneous pigmentary dermatoses of unknown etiology. In the present study, the leukocyte infiltrates in the affected skin of EDP and vitiligo patients were studied using the avidin-biotin (ABC) immunoperoxidase technique and monoclonal antibodies which recognise the following mononuclear cell subgroups: T-suppressor/cytotoxic (CD8-Leu-2), T-helper (CD4 = OKT4), T-suppressor + macrophages (Leu-15), Pan T (CD3 = Leu-4), macrophages (Leu-M3) and Langerhans cells (CD1 = Leu-6), and other cellular markers such as Ia antigens and the Interleukin-2 receptor (CD25 = TAC). The immunocytochemical analysis showed a selective accumulation of CD3+, CD8+, Leu-15-, T-cytotoxic cells in the epidermis of both EDP and early lesions of vitiligo. In addition, an increase in the number of epidermal Langerhans cells (CD1+) was observed in some cases of EDP and vitiligo. The CD4/CD8 ratios in affected and uninvolved skin for both disorders were not significantly different, although values lower than unity were only observed in the infiltrates of affected skin. Ia antigen positivity was observed in the dendritic cells of the dermis and epidermis, as well as in most of the lymphoid cells within the infiltrates for both diseases. Macrophages (Leu-M3) in EDP dermal infiltrates were generally found adjacent to extracellular melanin pigment. Lymphocytes expressing TAC (CD25) surface antigens were also present in the dermal infiltrates. These morphological observations suggest a possible immune cell participation in the dyschromia of such cutaneous disorders.     

The CD4 molecule transmits biochemical information important in the regulation of T lymphocyte activity

The role of the CD4 molecule in the transmission and regulation of the biochemical signals involved in T cell activation was investigated using an anti-CD4 monoclonal antibody termed 6B10. 6B10 immunoprecipitated the 55-kDa CD4 molecule and detected an epitope of CD4 that overlapped with that detected by OKT4A, B, and D. 6B10, 6B10 Fab fragments and recombinant HIV envelope glycoprotein (gp120) induced calcium mobilization in PBMC. 6B10 stimulation also resulted in calcium mobilization in murine L cells expressing transfected CD4 gene products, indicating that CD4-mediated calcium mobilization occurred independently of the CD3/T cell receptor (TCR) complex. 6B10 induced a phosphatidylinositol response, but the response resulted in reduced inositol phosphate production compared to levels obtained using OKT3. Though 6B10 caused calcium mobilization and a phosphatidylinositol response, 6B10 did not induce DNA synthesis. The amount of inositol phosphates produced by 6B10 may be below the threshold necessary for cell cycle progression. We hypothesized that 6B10-mediated calcium mobilization is important in the regulation of T cell proliferation. 6B10, but not 6B10 Fab fragments, inhibited OKT3-induced DNA synthesis. Furthermore, 6B10 but not 6B10 Fab fragments inhibited OKT3-induced calcium mobilization, suggesting that crosslinking of CD4 may be an important factor determining whether signals result in both the up- and down-regulation of CD3/TCR complex function. The implication of this work is that signals generated via the CD4 molecule are important in the regulation of T cell function and that the signals generated as a result of HIV gp120 binding to CD4 can contribute to the mechanism by which HIV inhibits T cell function.