Stimulation in primary MLR caused by a PLT defined non-HLA-D/DR determinant, EP1

The influence in primary mixed lymphocyte culture reaction (MLR) of a primed lymphocyte typing (PLT) defined non-HLA-D/DR determinant, EP1, was investigated. In primary MLR between HLA-D/DR compatible lymphocytes, the response of the lymphocytes from 14 EP1-negative HLA-D/DR heterozygous individuals towards two EP1-positive homozygous typing cells (HTCs) was on an average approximately 35% higher than the response towards two EP1-negative HTCs ( P < 0.01). The strength of the MLR between lymphocytes from 25 EP1-negative and 10 EP1-positive individuals matched for two HLA-D/DR antigens was investigated. The average responses of EP1-negative lymphocytes against EP1-positive lymphocytes were approximately 40% higher than the average responses against EP1-negative lymphocytes ( P < 0.01). These data indicate that the PLT defined determinant EP1 causes stimulation in primary MLR.     

Efficient Presentation of House Dust Mite Allergen Der p 2 by Monocyte-Derived Dendritic Cells and the Role of β2 Integrins

Monocyte-derived dendritic cells (MDDC) are potent antigen-presenting cells (APC). The APC functions of MDDC in allergy were examined. MDDC presentation of the major house dust mite allergen Der p 2 resulted in 4–12-fold higher T-cell proliferation and markedly higher IFN-γ and IL-5 production than PBMC cultures. Comparable T-cell proliferation was obtained with 10-fold fewer MDDC than purified monocytes. MDDC cultured from adherent cells, or CD14⁺, CD11b⁺ or Percoll-purified monocytes were comparable in presenting soluble Ag, and in stimulating allogeneic MLR. Importantly, MDDC presentation of Der p 2 resulted in both Th1 and Th2 stimulation, although MDDC are known to produce high levels of IL-12 and stimulate biased Th1 responses. The basis for the potent APC function of MDDC was further examined. MDDC were found to be highly phagocytic. Immunoprecipitation studies showed markedly elevated ICAM-1 expression but > 10-fold reduction in LFA-1 expression on MDDC compared with monocytes. Monoclonal antibody (MoAb)-blocking experiments showed that ICAM–LFA-1 interaction was essential for MDDC stimulation of Der p 2-specific T-cell proliferative responses. These results show that the use of MDDC as APC provides a simple, sensitive and versatile method for detecting T-cell responses to allergens and that the strong phagocytic capability and the increased ICAM-1 expression of MDDC contribute significantly to their Ag presentation potency.     

Intercellular adhesion molecule-3 is the predominant co-stimulatory ligand for leukocyte function antigen-1 on human blood dendritic cells

Dendritic cells (DC) are potent stimulators of primary T lymphocyte responses to foreign antigen. The initial DC-T lymphocyte interaction involves the binding of the adhesion molecule leukocyte function antigen-1 (LFA-1; CD11a/CD18) on the T lymphocyte to an intercellular adhesion molecule (ICAM) on the DC. Although blood and tonsil DC express ICAM-1 (CD54) and ICAM-2 (CD102) on their surface, anti-ICAM-1 and anti-ICAM-2 monoclonal antibodies (mAb) have little inhibitory activity on the DC-stimulated mixed leukocyte reaction (MLR). We therefore examined the expression of the more recently identified LFA-1 ligand, ICAM-3 (CD50), in comparison to ICAM-1 and ICAM-2 on blood DC and sought a functional role for ICAM-3 in DC-mediated T lymphocyte responses. Resting blood DC expressed significantly more ICAM-3 than ICAM-1 or ICAM-2 as assessed by flow cytometry. Treatment of resting DC with interferon-γ led to increased expression of ICAM-1; however, ICAM-2 and ICAM-3 levels remained relatively constant. Solid-phase recombinant chimeric molecules ICAM-1-, ICAM-2- and ICAM-3-Fc were able to co-stimulate CD4⁺ T lymphocyte proliferation in conjunction with suboptimal solid-phase CD3 mAb 64.1. However, the anti-ICAM-3 mAb CAL 3.10 inhibited a DC-stimulated MLR to a greater extent than anti-ICAM-1 or anti-ICAM-2 reagents and appeared to act by blocking the DC ICAM-3- T lymphocyte LFA-1 interaction. As ICAM-3 is the predominant LFA-1 ligand on resting blood DC, we postulate that DC may utilize ICAM-3 for initial DC-T lymphocyte interactions, and that ICAM-1, which is up-regulated upon DC activation, and/or ICAM-2, may contribute to DC migration or later phases of the T lymphocyte activation process.     

Efficient presentation of house dust mite allergen Der p 2 by monocyte-derived dendritic cells and the role of beta 2 integrins

Monocyte-derived dendritic cells (MDDC) are potent antigen-presenting cells (APC). The APC functions of MDDC in allergy were examined. MDDC presentation of the major house dust mite allergen Der p 2 resulted in 4-12-fold higher T-cell proliferation and markedly higher IFN-gamma and IL-5 production than PBMC cultures. Comparable T-cell proliferation was obtained with 10-fold fewer MDDC than purified monocytes. MDDC cultured from adherent cells, or CD14+, CD11b+ or Percoll-purified monocytes were comparable in presenting soluble Ag, and in stimulating allogeneic MLR. Importantly, MDDC presentation of Der p 2 resulted in both Th1 and Th2 stimulation, although MDDC are known to produce high levels of IL-12 and stimulate biased Th1 responses. The basis for the potent APC function of MDDC was further examined. MDDC were found to be highly phagocytic. Immunoprecipitation studies showed markedly elevated ICAM-1 expression but > 10-fold reduction in LFA-1 expression on MDDC compared with monocytes. Monoclonal antibody (MoAb)-blocking experiments showed that ICAM-LFA-1 interaction was essential for MDDC stimulation of Der p 2-specific T-cell proliferative responses. These results show that the use of MDDC as APC provides a simple, sensitive and versatile method for detecting T-cell responses to allergens and that the strong phagocytic capability and the increased ICAM-1 expression of MDDC contribute significantly to their Ag presentation potency.     

The Immunodeficiency of Bone Marrow-Transplanted Patients

Lymphocytes from 21 patients sampled 1-6 months after bone marrow transplantation (BMT) were tested for functional suppressor activity against marrow-donor lymphocytes in the lymphocyte transformation test. Suppression of donor responses to allogeneic (i.e. mixed lymphocyte reaction, MLR) and antigenic stimulation by irradiated (7600 rad) post-BMT cells was observed in about two-thirds of the combinations tested (N = 20 and N = 9). The suppression of donor MLR and antigen responses ranged between 5-52% and 10-46%, respectively. Irradiated post-BMT cells significantly suppressed donor responses to suboptimal concentrations of phytohaemagglutinin (PHA) (median suppression: 28%; P less than 0.05; N = 7) and concanavalin A (Con A) (median suppression: 31%; P less than 0.05; N = 6). A clearly suppressive effect of post-BMT cells was observed when the ratios of CD4+/CD8+ post-BMT cells were lower than 0.5 (P less than 0.01). In three experiments, the depletion of the CD8- but not of the CD4-positive subset abrogated the suppression of the donor MLR by post-BMT cells. The suppression by post-BMT cells (irradiated) of MLR and mitogen responses was comparable whether the responding cells were derived from the donor or from HLA-DR-incompatible, unrelated individuals. The proliferative capacity of post-BMT cells compared to that of donor cells was assayed in the MLR with unrelated, HLA-DR-incompatible stimulator cells. A significantly decreased proliferative capacity (median 20% of that of donor cells) was found (P less than 0.01; N = 16). A weak inverse correlation (P less than 0.05; N = 16) between the proliferative and the suppressive capacity of post-BMT cells in the MLR was observed. These findings indicate that the decreased proliferative capacity upon mitogen, antigen, and alloantigen stimulation observed in most patients within 1-6 months after BMT may be partly due to non-specific suppression by CD8+ cells.     

Regulation and Functional Involvement of Distinct Determinants of Leucocyte Function-Associated Antigen 1 (LFA-1) in T-Cell Activation In Vitro

The expression of leucocyte function-associated antigen 1 (LFA-1) was studied by immunofluorescence method on human peripheral blood mononuclear cells (PBMC) stimulated by the phytohaemagglutinin lectin (PHA). Monoclonal antibodies (MoAb) Mas 191c, IOT18 (directed against the beta-chain, 95 kDa, CD18) and IOT16, SPVL7, MHM24 (identificating the alpha-chain, 180 kDa, CD11a) were used, defining the 'CD11a/CD18' antibody panel. By means of cross-linking or competitive experiments, we showed that these antibodies recognized at least four distinct and spatially distant domains on the LFA-1 molecule. Immunofluorescence analysis revealed that the up-regulation of LFA-1 expression was a late event, similar to the expression kinetics of the HLA DR and CD38 molecules, and followed the appearance of CD25 and CD71 molecules. Moreover, it was established that the LFA-1 up-regulation required mRNA and protein synthesis. Functional activity comparison of the different anti LFA-1 MoAb showed that the CD11a MoAb significantly inhibited the proliferation of lymphocytes stimulated by the phytohaemagglutinin to various extents, as the LFA-1 alpha determinant identified. By contrast, the CD18 MoAb did not influence strongly this cell process. We observed only a dim inhibitory effect with the CD18 MoAb recognizing an epitope common or very close to an LFA-1 alpha determinant. These results suggested that the LFA-1 antigen was important, at a molecular level, in the regulation of T-cell activation.     

Acquisition of Interleukin-5 Secretion by Human Naive T-Helper Cells is Regulated by Distinct Signals from both the T-Cell Receptor/CD3 Complex and CD2

We developed a human naive T-helper (Th) cell differentiation model with anti-CD3 monoclonal antibody (MoAb), using a B-cell line as source of costimulation. In this system, we examined the contribution of the T-cell receptor (TCR)/CD3-derived signals and that of lymphocyte function-associated antigen-1 (LFA-1) and CD2 in regulating Th-cell subset differentiation. We found that lowering the level of anti-CD3 MoAb decreased tumour necrosis factor-α (TNF-α) production, while increasing secretion of the Th2 cytokines, interleukin-5 (IL-5) and interleukin-13 (IL-13). Secretion of interferon-γ (IFN-γ) was not influenced by the strength of the anti-CD3 signal. Under conditions where Th0 cells are generated, co-culture with anti-CD2 F(ab')₂ MoAb led to the generation of Th cells that secreted 30–35% less IL-5, while not affecting secretion of IFN-γ or granulocyte–macrophage colony-stimulating factor (GM-CSF). By contrast, anti-CD18 MoAb F(ab')₂ inhibited IFN-γ and GM-CSF levels only in the primary stimulation, but did not affect cytokine levels after restimulation. Neither anti-CD2 nor anti-CD18 F(ab')₂ MoAbs could alter cytokine secretion profiles of peripheral blood-derived memory/effector Th cells. Our results indicate that acquisition of IL-5 secretion capability by Th cells is regulated mainly by signals transduced by the TCR/CD3 complex and by the presence of interleukin-4 (IL-4), while the CD2/LFA-3 pathway plays an additional, but minor, role. These regulatory CD2-derived signals, however, are distinct from those generated by the TCR/CD3 complex.     

The Stimulation of T Cells with Anti-CD2 Monoclonal Antibodies Facilitates the Induction of Polyclonal B-Cell Responses but does not Enhance the Activation of Antigen-Specific B Cells

In this report we compare the effect of stimulation of peripheral mononuclear cells (PBMC) by using two monoclonal antibodies (MoAb) directed against the CD2 receptor on T cells or by using autologous erythrocytes (E) which express on their surface lymphocyte function-associated antigen 3 (LFA3), a natural ligand for CD2. The addition of autologous erythrocytes to pokeweed mitogen (PWM)-stimulated PBMC results in the enhancement of polyclonal immunoglobulin synthesis and of antigen-specific B-cell responses. Because B cells lack the CD2 molecule, it can be concluded that their enhanced activity is a consequence of the delivery of activating signals by activated T lymphocytes. When PBMC cultures were stimulated with a pair of anti-CD2 MoAb (Leu5b and VIT13) we were able to induce polyclonal immunoglobulin synthesis, particularly IgM, in cultures supplemented with interleukin 2(IL-2). Specific responses to tetanus toxoid (TT) and keyhole limpet haemocyanin (KLH) were also enhanced by the addition of autologous E to PWM-stimulated PBMC. Significant anti-TT responses were observed in cultures stimulated with E + TT + IL-2. In contrast, stimulation of PBMC with VIT13 + Leu5b + IL-2 + antigen was not effective in inducing anti-TT antibody and only weakly effective in inducing anti-KLH antibodies. Replacing Leu5b by anti-CD3 had no effect on the induction of specific antibody responses; in contrast, replacement of Leu5b by E enhanced anti-TT antibody production while the effect on polyclonal production of IgM was minimal. Therefore, it appears that the signal delivered by the association of CD2 with LFA3 is a better potentiating signal for specific B-cell responses than the signal delivered by pairs of MoAb to different epitopes of CD2 or to CD2 and CD3 epitopes.     

Functional Epitope Analysis of the Human CD 11 a/CD 18 Molecule (LFA-1, Lymphocyte Function-Associated Antigen 1) Involved in HIV-1-Induced Syncytium Formation

After binding to the CD4 receptor, the human immunodeficiency virus 1 (HIV-1) may enter the T cell and induce the formation of multinucleated giant cells (syncytia). As well as the CD4 molecule, other molecules, such as the lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18) have been shown to be involved in HIV-1-mediated cell fusion. This study was designed to define regions on the human CD11a/CD18 molecule important for the HIV-1-induced syncytium formation. A CD11a/CD18 MoAb panel discriminating at least five distinct and spatially distant domains on the LFA-1 molecule was used. Comparison of the functional activity of different MoAbs demonstrated that all epitopes of the LFA-1 molecule were not of equal importance in HIV-1-induced syncytium formation between H9.III cells chronically infected with HIV-1 and uninfected CD4+ SupT1 cells. We also demonstrated that CD11a/CD18 MoAbs inhibit syncytia formation only at the level of the uninfected SupT1 cells, suggesting that the LFA-1 molecule expressed on SupT1 cells interacts with ligand(s) expressed on the infected H9.III cells. Two potential LFA-1 receptors on the H9.III cells were tested: the ICAM-1 molecule (intercellular adhesion molecule 1, CD54) and the HIV-1 transmembrane glycoprotein 41 (gp41). A CD54 MoAb (84H10) partially inhibited syncytia formation, thus demonstrating the involvement of the ICAM-1 molecule in the HIV-1-mediated cell fusion. However, the CD11a/CD18 MoAbs do not inhibit binding of the viral envelope glycoprotein gp41 to the cell surface, irrespective of the MoAb concentration used. Although we have not been successful in identifying all candidate fusion receptors for the LFA-1 molecule, these data suggest that some LFA-1 regions are important for syncytium formation and, therefore, in the cell-to-cell transmission of virus and in the spread of infection.     

Accessory Signals in T-T Cell Interactions Between Antigen- and Alloantigen-Specific, Human Memory T Cells Generated in Vitro

The potential of activated HLA class II-positive T cells as antigen-/alloantigen-presenting cells remains controversial. In our model system we use in vitro-primed, HLA class II-specific T cells of the memory T-cell phenotype, CD4+, CD29+ (4B4+), and CD45RO+ (UCHL-1). We have previously shown that alloactivated, HLA class II-positive T cells (Ta) are unable to stimulate proliferative responses in naive and primed allospecific T cells when 'back-stimulation' is avoided. The explanation of this feature of Ta is unknown, but it is due neither to suppression nor to insufficient HLA class II expression. Accordingly, we investigated the possibility that Ta have a deficient expression of accessory signals critical for the induction of proliferative T-cell responses. We found that (1) non-mitogenic concentrations of phorbol myristate acetate (PMA) in combination with either rIL-4, a CD28-reactive MoAb (Kolt-2), or a calcium ionophore (A23187) enabled Ta to elicit alloantigen-specific memory T-cell responses and to present purified protein derivative (PPD) to PPD-specific T-cell lines. The addition of irradiated, Epstein-Barr virus-transformed B-cell lines (EBV-LCL) (but not their supernatants) had a similar but less pronounced effect; (2) MoAb directed against HLA class II, CD25 (IL-2R), CD2, CD4, CD11a (LFA-1), or CD45RO molecules inhibited these responses; (3) PMA was required within the first hour of culture in order to induce optimal alloantigen-specific T-cell activation, while rIL-4 was fully effective when added after 20-44 h of culture; (4) incubation for 20 h of Ta with rIL-4 plus PMA markedly up-regulated CD54 expression on the Ta, and IL-4 seemed to potentiate the effect of PMA on the CD54 expression. In conclusion, the present data indicate that the inability of Ta to elicit (allo)antigen-specific, proliferative T-cell response is due to a lack of critical accessory signals. Up-regulation of CD54 was not sufficient for Ta to stimulate proliferative responses. Neither cytokines (IL-1, IL-6, and others) nor triggering of CD2 epitopes (T11.2 and T11.3) by soluble MoAb or solid phase support by MoAb against a number of accessory molecules provided the necessary signals. Thus, our data indicate that other, as yet unknown, signalling pathways play a key role in antigen- and alloantigen-specific T-T interactions. These pathways still need to be identified.     

Ivermectin-facilitated immunity in onchocerciasis. Reversal of lymphocytopenia, cellular anergy and deficient cytokine production after single treatment.

A longitudinal investigation has been conducted into the cell-mediated immune responses of onchocerciasis patients after a single-dose treatment with ivermectin. Untreated patients tested for delayed cutaneous hypersensitivity (DCH) to seven recall antigens showed lower responses than infection-free control individuals (P less than 0.01), but 6 and 14 months after treatment DCH reactions increased to similar levels to those seen in the controls. The in vitro cellular reactivity to Onchocerca volvulus-derived antigen (OvAg) was reduced in untreated patients as compared with controls, and the lymphocyte blastogenic responses to OvAg and streptolysin-O clearly improved up to 14 months after treatment. Peripheral blood mononuclear cells (PBMC) from untreated patients produced IL-1 beta, tumour necrosis factor-alpha (TNF-alpha) and IL-6 in response to mitogenic stimulation with phytohaemagglutinin (PHA), only low levels of IL-1 beta, IL-2 and TNF-alpha in response to OvAg, but higher amounts of IL-4 and interferon-gamma (IFN-gamma) in response to OvAg than control individuals. After ivermectin treatment, the OvAg-induced production of IL-1 beta and TNF-alpha increased significantly 1 and 14 months after treatment. The PHA-induced production of IL-2 and IL-4 increased 1 month after treatment and remained significantly elevated until 14 months after treatment, whereas the OvAg-specific secretion of IL-2, IL-4 and IFN-gamma did not change after ivermectin treatment. Flow cytometric analysis of lymphocyte-subsets in the peripheral blood of untreated patients revealed a relative and absolute (P less than 0.01) diminution of CD4+ cells and a significantly smaller CD4+/CD8+ cell ratio as compared with controls. By 4 weeks after treatment and thereafter, CD4+ T cells increased relatively and absolutely (P less than 0.01); likewise there was an absolute increase in T-helper-inducer cells (CD4+CD45RO+) and a temporarily improved CD4+/CD8+ cell ratio (P = 0.001). The expression of the low-affinity receptor for IgE (CD23) on total lymphocytes decreased from 14% to 7% by 14 months after treatment. The CD8+ cells and CD3+TCR gamma delta + cells were higher in patients than in controls and both remained elevated until 14 months after treatment. These results suggest a distinctly improved cellular immunity in human onchocerciasis that was facilitated by ivermectin therapy.     

Study of induction of activation of human peripheral blood mononuclear cells with a non-activating form of anti-CD3 MoAb in autoimmune thyroid disease (AITD).

Anti-CD3 (OKT3) MoAb is a mitogenic agent which activates lymphocytes. We have studied the effects of murine anti-human OKT3 MoAb (IgG1) alone or in combination with IL-2, human thyroglobulin (Tg) and thyroperoxidase (TPO) antigens on the proliferation of whole peripheral blood mononuclear cells (PBMC) (including monocytes) or subtypes (T, CD4+, CD8+, B) as measured by tritiated thymidine (3H-TdR) incorporation. B cell differentiation was studied by measuring numbers of IgG-secreting cells and specific anti-TPO/anti-Tg-secreting cells by SPOT ELISA. PBMC or lymphocyte subtypes, obtained from 45 patients with Hashimoto's thyroiditis (HT), 40 Graves' disease (GD) and 51 normal controls were cultured in 96 microtitre plates for 6 days in the presence of OKT3 MoAb at final concentrations 25-250 ng/ml, IL-2 15 U/ml, Tg and TPO (1 micrograms/ml). Then cultures were pulsed with 0.2 microCi 3H-TdR/well and incorporation was measured after 18 h. IgG and anti-TPO/Tg-secreting cells were detected at 7 days. Higher proliferative responses from whole PBMC preparations in response to any of the combinations including OKT3 MoAb were observed in the HT preparations, while the basal values were the lowest. IL-2 alone increased these responses markedly, but equally in all groups. IL-2 in combination with OKT3 had an additive effect on proliferation, with higher responses in HT. Tg and TPO antigens did not change these responses. Most HT preparations responded with their maximum proliferation to the lowest concentration of OKT3 MoAb (25 ng/ml), whereas in GD and control preparations of PBMC these responses were shifted to higher concentrations (250 ng/ml); even with those, proliferation was not so enhanced in controls when compared with HT and GD preparations. In contrast, the proliferative responses of T cells alone and subpopulations of CD8+ suppressor/cytotoxic cells were decreased in HT preparations compared with controls. Monocytes were necessary for proliferation. In the subpopulation of B cells (> 95% pure) and CD4+ helper/inducer cells, differences did not reach significance. In spite of the effect on proliferation, OKT3 MoAb only mildly but significantly increased the numbers of IgG-secreting cells in HT and GD preparations and did not stimulate synthesis of specific antibodies. Our data suggest that the increased proliferative responses of whole PBMC to OKT3 MoAb in HT preparations might be due to insufficient activation of T suppressor/cytotoxic cells.     

NK cells inhibit T-cell responses: LFA3+ but not LFA3- T-cell responses are suppressed.

There is increasing evidence that natural killer (NK) cells have immunoregulatory effects in addition to their ability to lyse tumour and virus-infected target cells. However, much of the evidence to date is based on the reported effect of adding relatively impure NK cell populations to various in vitro cultures, and the effect on T cells has been contradictory. Here we report the inhibitory effect of highly purified CD16+ NK cells on mixed lymphocyte reaction (MLR), using unseparated peripheral blood mononuclear cells (PBMC) and purified T cells as responders. Marked inhibition was observed (up to 75%) which was proportional to the number of CD16+ cells present, and was abrogated by ultraviolet irradiation. In contrast, the addition of CD16+ cells had no effect on the proliferative responses of five CD4+ anti-DR1 alloreactive T-cell clones. To test the relative sensitivity of previously primed versus virgin T cells to NK cell-mediated inhibition, freshly isolated T cells from PBMC were separated into LFA3+ (memory) and LFA3- (virgin) populations. CD16+ cells caused inhibition of proliferation of LFA3+ but not LFA3- cells in an MLR. In addition, the recall response of T cells to influenza was inhibited. These results further illustrate the regulatory potential of CD16+ NK cells, and suggest that previously primed cells are more susceptible to NK-mediated inhibition. However, activated (rather than resting) cells may escape regulation.     

Requirement for cell-to-cell contact for the immunosuppressive activity of human alveolar macrophages

The ability of alveolar macrophages (AM) obtained by bronchoalveolar lavage of healthy volunteers to suppress T lymphocyte responses to the mitogen phytohemagglutinin (PHA) in vitro was investigated. AM but not monocytes (MN) inhibited responses of peripheral blood mononuclear cells (PBMC) to PHA as measured by incorporation of [3H]thymidine [( 3H]TdR) and interleukin-2 (IL-2) expression. Supernatants of AM generated for various periods and with various concentrations of cells did not, however, inhibit PBMC responses to PHA. To examine the role of cell contact in the inhibitory activity of AM, AM or MN were added to PBMC in 6-well plates either directly (in co-culture) or separated by a 0.45-micron filter. MN did not inhibit PBMC blastogenic responses under either condition. AM at a 1:2 ratio with PBMC inhibited blastogenesis by 75 +/- 11% (mean +/- SD, n = 3, P less than 0.01) when cultured directly with PBMC but had no inhibitory effect on blastogenesis when physically separated from target PBMC. AM in co-culture with PBMC also inhibited PHA-stimulated IL-2 production by 70% but did not inhibit IL-2 production when AM were separated from PBMC in dual chambers. To assess the role of the cell surface in the inhibitory activity of AM, AM and MN were fixed with 2% paraformaldehyde. Neither fixed nor unfixed MN inhibited PBMC blastogenic responses, but both fixed and unfixed AM inhibited responses similarly (77 to 95%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of CD8+ T cells by allogeneic class II-deficient B-cell lines derived from patients with bare lymphocyte syndrome

The major histocompatibility complex (MHC) class II antigens (Ags) are known to carry the major stimulating determinants of the primary mixed lymphocyte reactions (MLR). We investigated the mechanism of generating HLA class I-directed alloreactive T-cells in primary MLR. With the use of class II-deficient EBV-transformed B-lymphoblastoid cell lines (B-LCLs) derived from patients with bare lymphocyte syndrome (BLS), we have demonstrated in the present study that class I disparity alone can trigger primary MLR in the absence of exogenous IL-2. The CD8+ T cells were primary MLR-responsive cells, and the CD4+ T cells seem to play no role in primary MLR when class II alloantigens are not involved in stimulation. Addition of autologous macrophages did not influence the primary MLR response. The primary MLR was completely blocked by anti-class I or anti-CD8 antibodies but not by anti-class II or anti-CD4 antibodies. The MLC-generated CD8+ T cells exhibited cytolytic activity as well as proliferative responses. The proliferative response of the CD8+ T cells was specifically directed against class I antigens, demonstrated by proliferative assays; and the helper-independent CD8+ T cells were generated only when the activation of CD4+ T cells did not occur. This observation suggests that functional recruitment of T-cell receptor (TCR) repertoire is under active regulation, and the suppression of CD8+ T-cell helper recruitment appears to be dictated by the CD4+ T-cell subset. Further analysis of the primed T-cell specificities showed that alloreactivity of the CD8+ T cells was mostly accounted for by the HLA-B Ags.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro effects of thymosin and lithium on lymphoproliferative responses of normal donors and HIV seropositive male homosexuals with AIDS-related complex

The in vitro effects of thymosin fraction 5 (TF5) and lithium chloride (LiCl) on the ability of peripheral blood mononuclear cells (PBMC) obtained from 37 normal male donors and 33 male patients with AIDS-related complex (ARC) to respond to alloantigenic stimulation (mixed leukocyte reaction, MLR) and to produce interleukin 2 (IL-2) in response to mitogens were studied. TF5 significantly increased MLR responses in normal donors (P less than 0.01) and in a group of 33 ARC patients with depressed cellular immunity (P less than 0.05). Similar effects were observed when LiCl was added to the MLR assays in both the normal and the ARC patient groups. Furthermore, TF5 and LiCl exhibited additive immunoenhancing properties. In 10 normal donors TF5 enhanced phytohemaggutinin (PHA)-induced IL-2 production as well as IL-2 production in response to pokeweed mitogen (PWM) (P less than 0.02). TF5-mediated enhancement of IL-2 production by PBMC obtained from ARC patients was observed in response to both mitogens, i.e., PHA and PWM. Additionally, LiCl increased PHA-induced IL-2 production in both normal subjects and ARC patients. LiCl and TF5 together had an additive effect in the enhancement of IL-2 production in both groups of subjects. Our data extend previous observations regarding the immunoregulatory activities of TF5 and LiCl and provide evidence that PBMC obtained from ARC patients have the potential to respond in vitro to these agents. The significance of these findings is discussed.     

Selective impairment of T lymphocyte activation through the T cell receptor/CD3 complex after cytomegalovirus infection.

Cytomegalovirus (CMV) infection is reported to cause transient immunosuppression in man. In this study we have analysed the effect of CMV on T lymphocyte function in 29 children diagnosed for acute CMV infection. Peripheral blood mononuclear cells (PBMC) obtained from the patients showed a significant specific impairment in their proliferative response to enterotoxins A and C1, to concanavalin A and to the anti-CD3 MoAb OKT3. The impaired responses were corrected with exogenous IL-2. In contrast, stimulation using phytohaemagglutinin, as well as activation signals delivered through the surface molecules CD26 or CD28, elicited normal proliferative responses in CMV PBMC. The results indicate that the T cell anergy associated with CMV infection is restricted to the T cell receptor/CD3 activation pathway.     

Interleukin 2 receptor expression by human blood lymphocytes after vaccination with pneumococcal polysaccharides.

Proliferative responses of unseparated peripheral blood mononuclear cells (PBMC) and blood T cells to recombinant interleukin 2 (rIL-2) were significantly increased 7-21 days after the vaccination with pneumococcal polysaccharides (PPS). In contrast, non-T cells expressed increased responsiveness to rIL-2 only on post-vaccination day 7. Analysis of the proliferative response to rIL-2 among lymphocyte subsets (CD4+Leu8+, CD4+Leu8-, CD8+Leu8+, CD8+Leu8-, CD20+) in cultures of unseparated PBMC revealed that the CD8+Leu8- T cells expressed increased responsiveness 7-14 days after vaccination, whereas neither CD4+ (Leu8+ and Leu8-) nor CD8+Leu8+ T cells showed significantly increased responsiveness after vaccination. The CD20+ B cells, like non-T cells, expressed increased responsiveness to rIL-27 days after the vaccination only. Expression of the 55 kD low-affinity interleukin 2 receptor (IL-2R, CD 25) on freshly isolated PBMC, as judged by direct fluorescence staining with a MoAb anti-55 kD chain, was low (less than 3%) and an increased expression of this receptor was not detected following vaccination. In contrast, binding of 125I-labelled IL-2 to freshly isolated PBMC increased following vaccination (day 7). Scatchard plot analysis revealed a modest increase in the expression of high-affinity IL-2R (Kd = 1-2 pM), whereas the increase in expression of the 75-kD, intermediate-affinity IL-2R (Kd = 300 pM) was more pronounced (from 195 to 295 (means) receptors per PBMC). It is concluded that, following vaccination with PPS increased IL-2R expression is induced on blood lymphocytes. These investigations suggest a role for T cells in the human immune response against PPS.     

Effects of cyclosporin A on in vitro lymphocyte response to autologous or allogeneic stimulation: influence of HLA phenotypes

In this study we investigated whether the interindividual variability of lymphocyte sensitivity to cyclosporin A (CsA) could be controlled by the HLA region. The models used were the in vitro primary and secondary autologous (AMLR) and allogeneic mixed lymphocyte (MLR) cultures of cells from 32 healthy subjects from our HLA reference panel. Our results show that CsA inhibited primary allogeneic MLR to a much greater extent than primary AMLR (-81 +/- 2% vs -38 +/- 8%, P less than 0.001). The same pattern was observed when cells harvested from CsA-treated primary cultures were rechallenged in secondary cultures with the original sensitizing stimulator cells (-40 +/- 6% vs -17 +/- 9%, P less than 0.05). No differences were observed in primary autologous and allogeneic cultures among responders of different HLA phenotypes. In contrast, the secondary responses did vary according to the HLA types: in secondary AMLR, CsA-priming did not lower, or even enhance, the proliferative responses of DR5+ and/or DR2+ lymphocytes (+7 +/- 13%), whereas it significantly lowered the responses of DR2-5- cells (-46 +/- 8%). In secondary MLR, lymphocytes proliferation was lowered by CsA-priming in all but DRW11(5)+ subjects (-45 +/- 7% vs +2 +/- 23%, P less than 0.05). It is concluded that the individual HLA phenotype influences the pattern of lymphocyte sensitivity to CsA.     

T lymphocyte anergy during acute infectious mononucleosis is restricted to the clonotypic receptor activation pathway.

The transient T cell anergy associated with acute infectious mononucleosis (IM) caused by the Epstein-Barr virus has been analysed in a sample of 14 IM children. Peripheral blood mononuclear cells (PBMC) obtained from IM patients showed a significant specific impairment in their proliferative response to both phytohaemagglutinin (PHA; P less than 0.05) and to an anti-CD3 MoAb (P less than 0.001), although both responses reached normal control levels by addition of a submitogenic dose of either phorbol myristate acetate (PMA) or recombinant IL-2 (rIL-2). In contrast, activation signals delivered through other surface molecules (CD2, CD28) or other transmembrane pathways (PMA plus a calcium ionophore) elicited normal or high proliferative responses in most IM PBMC. In a group of five patients tested, the synthesis of IL-2 by IM PBMC in the presence of PMA was impaired when PHA or anti-CD3 was used as stimulus, but it reached normal levels with anti-CD2 or ionophore. Lastly, PHA failed to induce IL-2 alpha receptor (IL-2R alpha) expression in IM PBMC from four tested patients, but the presence of PMA completely corrected this defect. Taken together, these results strongly suggest that the T cell anergy associated with acute IM is due to a T cell receptor (TCR)-specific impairment in the induction of genes involved in T cell proliferation (including those coding for IL-2 and IL-2R alpha) upon membrane signalling to otherwise normal T lymphocytes, since CD2, CD28 and certain transmembrane activation pathways are uncoupled from CD3 in these particular pathological conditions (and perhaps in most in vivo situations). This and other similar experimental approaches to transient secondary immunodeficiencies may help to unravel the physiopathological role of different surface molecules in T cell activation.