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We investigated interleukin-10 (IL-10) production in freshly isolated mononuclear cells and purified T cells in response to stimulation with monoclonal antibodies (mAb) recognizing CD3, CD2 and CD28, or with the bacterial products Staphylococcus aureus cells (SAC), staphylococcal enterotoxin (SEA) and lipopolysaccharide (LPS). IL-10 production was compared with that of IL-2, IL-4 and interferon-γ (IFN-γ). Similar to the other cytokines, in peripheral blood mononuclear cells (PBMC) from adult donors the highest IL-10 levels were produced in response to CD2 plus CD28 stimulation, within 72–96 hr of stimulation. Levels of IL-10 in response to CD2 plus CD28 stimulation (1·9 ± 1 ng/ml) exceeded those in response to SEA (0·25 ± 0·16 ng/ml), SAC (0·43 ± 0·42 ng/ml), or LPS (0·19 ± 0·14 ng/ml) stimulation. With adult purified T cells, high levels of IL-10 and IL-4 were measured following CD3 plus CD28 stimulation, and the amounts of both T-helper type-2 (Th2) cytokines decreased following the addition of phorbol myristate acetate (PMA), whereas the synthesis of the Thl cytokines IL-2 and IFN-γ was enhanced. When PBMC were stimulated with a CD3 mAb and different other cytokines were added, strong enhancement of IL-10 production was seen upon the addition of IL-2, IL-4, IL-7, IL-12 and IFN-γ, whereas inhibition was found with transforming growth factor-β1 (TGF-β1). These data illustrate that in freshly isolated PBMC large amounts of IL-10 can be induced rapidly by appropriate mAb stimulation, and that even in freshly isolated cells IL-4 and IL-10 show signs of parallel regulation. [ABSTRACT FROM AUTHOR]

While it is clear that the β subunit of interleukin-2 receptor (IL-2R) plays a pivotal role in IL-2 induced signal transduction, the function of the α subunit, other than modulating the association rate of IL-2, is still unknown. It has been reported that the interaction between ID2 and the IL2Rα, subunit of several IL-2-dependent murine T-cell lines may result in a negative regulatory signal. To confirm this finding, we investigated the effect of an anti-IL-2Rα antibody, CD25-8D8, on the proliferative response of human peripheral blood lymphocytes. Lymphocytes from purified protein derivative (PPD)-positive donors were cultured with PPD and various concentrations of CD25-8D8 for up to 9 days, and [³H]thymidine uptake was measured. Whereas the proliferative response of human lymphocytes to PPD was suppressed by high concentrations of CD25-8D8, subinhibitory amounts of CD25-8D8 enhanced lymphocyte proliferation by 3.5-fold (range 2.26.2-fold) on the second day after maximal [³H]thymidine uptake had occurred, By itself, CD258D8 could not induce proliferation of washed 5-day PPD-activated lymphocytes during reculturing; instead, growth enhancement by CD25-8D8 was dependent on the presence of PPD-activated culture supernatant or moderate levels of exogenous IL-2. The enhancing effect of anti-IL-2Rα antibody, observed in both murine and human systems, reinforces the possibility that binding of IL-2 to the IL-2Rα, chain plays a negative regulatory role in signal transduction. [ABSTRACT FROM AUTHOR]

CD43 is a cell-surface sialoglycoprotein which is selectively expressed on lympho-haemopoietic cells. We studied the effects of three CD43 antibodies (6E5, 6F5 and 10G7) on human neutrophils and found that all three monoclonal antibodies (mAb) induced significant homotypic adhesion involving more than 50% of cells. Monovalent Fab fragments of CD43 mAb had no such effect but became equally effective upon cross-linkage with F(ab')2 sheep anti-mouse immunoglobulin (Ig) antibodies. The homotypic adhesion induced by CD43 antibodies was dependent on divalent cations, energy, temperature and an intact cytoskeleton, but not on de novo protein synthesis. Homotypic adhesion could be inhibited by mAb to CD11b, CD18 and CD54, indicating an involvement of the β2 integrin cyto-adhesion pathway. Additionally, oxidative burst formation was observed with intact CD43 mAb. No such effect was seen with monomeric or cross-linked Fab fragments. This, together with the observation that burst formation unlike adhesion induction could be completely abolished with FcγRII, but not with FcγRIII antibody fragments, suggests that in burst induction, heterologous cross-linkage with FcγRII is involved. A Ca2+ increase with CD43 antibodies was not detectable. Adhesion induction was unaffected by H7, chelerythrin, staurosporine or lavendustin A, but was completely ablated by sphingosine and herbimycin A. This suggests an involvement of tyrosine kinases but not of protein kinase C in the signal transduction cascade leading to homotypic adhesion. CD43 mAb-induced burst formation differed from adhesion induction in that it could be additionally inhibited with staurosporine and lavendustin A. [ABSTRACT FROM AUTHOR]

HLA class I molecules on activated T cells are expressed as heterodimers associated with β2microglobulin (β2-m) and also β2-m-free HLA class I α-chains. Mechanisms leading to the expression of the activation associated β2-m-free HLA class I α-chains are poorly defined, however. Upon enzymatical removal of HLA class I α-chains on activated T cells, re-expression is observed within minutes upon reculture, reaching half-maximal levels within 1 hr. This process is independent of de novo protein synthesis and of export of newly synthesized proteins. Inhibition of the formation of coated pits by potassium depletion of cells abrogated the re-expression of HLA class I α-chains, suggesting that recycling events of HLA class I heterodimers via endosomal compartments are required for the generation of monoclonal antibody LA45-reactive α-chains. Furthermore, the rate of α-chain generation seems to be governed by the amount of cell surface-expressed HLA class I heterodimers. Taken together these findings suggest that β2-m-free HLA class I α-chains are generated during the process of class I heterodimer recycling. [ABSTRACT FROM AUTHOR]

Some (VIM12, Leu-l5, 5A4.C5), but not all, Mac-l-specific monoclonal antibodies (mAb) induced a clear respiratory burst in unprimed monocytes but not in unprimed polymorphonuclear leucocytes (PMN). We showed that this monocyte stimulation occurred via formation of Mac-I mAb-FcγRl or Mac-I mAb FcγRII complexes, as human monomeric IgG1 could completely block the respiratory burst induced by the routine IgG2a subclass anti-Mac-I mAb Leu-15 and the FcγRII-specific mAb IV.3 inhibited respiratory burst formation by IgGI subclass anti-Mac-I mAb V1MI2 and 5AA.C5, respectively. F(ab')2 fragments of mAb VIM12 did not stimulate. This association between Mac-I and FcγRII may be due to a near spatial association between these molecules in monocytes, as we observed partial inhibition of FITC-labelled anti-FcγRII mAb IV.3 binding after prior incubation with mAb VIMI2. If monocytes were preincubated with mAb IV.3 or aggregated IgG, there was partial inhibition of mAb VIM12 binding. The non-stimulating anti-Mac-1 mAb (JML.H11,44, OKM1, LM2/1, Mol) did not show any significant competition with mAb IV.3 binding to FcγRII. Both non-stimulating CD18-specific mAb, however, showed strong competition with mAb IV.3 binding to FcγRII. On unprimed PMN, the situation was different. No Mac-l-specific mAb induced a respiratory burst and there was no competitive inhibition between anti-Mac-1 mAb and antibodies binding to FcγRII. In interferon-γ (IFN-γ)-primed PMN, however, we observed a functional association between Mac-1 and FcγRI as IgG2a subclass mAb Leu-15 induced a respiratory burst which could be inhibited by monomeric human IgG1, as observed in monocytes. However, no other anti-Mac-1 mAb was able to induce a respiratory burst in IFN-γ-primed PMN. Therefore, a similar signal transducing capability may exist between Mac-l and FcγRI on both monocytes and PMN, despite a different relationship between Mac-1 and FcγRII on these cell populations. As no Mac-I β-chain-specific (CDI8) mAb were able to induce a respiratory burst in monocytes, despite being able to interact with FcγR via their Fc regions, as detected by competition with mAb IV.3 for binding to FcγRII, we conclude that intracellular signalling via Mac-I mAb-FcγRII complexes requires the α-chain. [ABSTRACT FROM AUTHOR]

The CD31 molecule is a leucocyte-surface glycoprotein of 130 kDa with homology to the immunoglobulin gene superfamily. In this study we report on the expression of CD31 on human T cells and demonstrate that it subdivides peripheral blood T lymphocytes into two novel subsets. CD31 is expressed by 38% of CD3+ lymphocytes. About 85% of CD313+ T cells display the CD45RA phenotype, 35% the CD45RO phenotype, 24% the CD4 phenotype and 72% the CD8 phenotype. There is also a correlation between CD31 expression and CD45RA expression in cord blood T cells; 89% of CD33+ cord blood cells express CD31, and most of them have the CD45RA phenotype. A discrepancy was found with thymocytes, which are positive for CD31 but negative for CD45RA. Stimulation of human T cells leads to down-regulation of CD45RA, while CD31 continues to be expressed. In functional studies, CD31 antibody binding to T lymphocytes does not lead to mobilization of intracellular calcium, proliferation or modulation of T-cell proliferation. [ABSTRACT FROM AUTHOR]

Co-receptors, being either co-stimulatory or co-inhibitory, play a pivotal role in T-cell immunity. Several studies have indicated that CD43, one of the abundant T-cell surface glycoproteins, acts not only as a potent co-receptor but also as a negative regulator for T-cell activation. Here we demonstrate that co-stimulation of human peripheral blood (PB) T cells through two distinct CD43 epitopes recognized by monoclonal antibodies (mAb) CD43-6E5 (T 6E5-act ) and CD43-10G7 (T 10G7-act ) potently induced T-cell proliferation. However, T-cell co-stimulation through two CD43 epitopes differentially regulated activation of nuclear factor of activated T cells (NFAT) and nuclear factor-κB (NF-κB) transcription factors, T-cell cytokine production and effector function. T 6E5-act produced high levels of interleukin-22 (IL-22) and interferon-γ (IFN-γ) similar to T cells activated via CD28 (T CD 28-act ), whereas T 10G7-act produced low levels of inflammatory cytokines but higher levels of regulatory cytokines transforming growth factor-β (TGF-β) and interleukin-35 (IL-35). Compared with T 6E5-act or to T CD 28-act , T 10G7-act performed poorly in response to re-stimulation and further acquired a T-cell suppressive function. T 10G7-act did not directly inhibit proliferation of responder T cells, but formed stable heterotypic clusters with dendritic cells (DC) via CD2 to constrain activation of responder T cells. Together, our data demonstrate that CD43 is a unique and polarizing regulator of T-cell function., (© 2016 The Authors. Immunology Published by John Wiley & Sons Ltd.)

HLA class I molecules on activated T cells are expressed as heterodimers associated with beta 2-microglobulin (beta 2-m) and also beta 2-m-free HLA class I alpha-chains. Mechanisms leading to the expression of the activation associated beta 2-m-free HLA class I alpha-chains are poorly defined, however. Upon enzymatical removal of HLA class I alpha-chains on activated T cells, re-expression is observed within minutes upon reculture, reaching half-maximal levels within 1 hr. This process is independent of de novo protein synthesis and of export of newly synthesized proteins. Inhibition of the formation of coated pits by potassium depletion of cells abrogated the re-expression of HLA class I alpha-chains, suggesting that recycling events of HLA class I heterodimers via endosomal compartments are required for the generation of monoclonal antibody LA45-reactive alpha-chains. Furthermore, the rate of alpha-chain generation seems to be governed by the amount of cell surface-expressed HLA class I heterodimers. Taken together these findings suggest that beta 2-m-free HLA class I alpha-chains are generated during the process of class I heterodimer recycling.

While it is clear that the beta subunit of interleukin-2 receptor (IL-2R) plays a pivotal role in IL-2-induced signal transduction, the function of the alpha subunit, other than modulating the association rate of IL-2, is still unknown. It has been reported that the interaction between IL-2 and the IL-2R alpha subunit of several IL-2-dependent murine T-cell lines may result in a negative regulatory signal. To confirm this finding, we investigated the effect of an anti-IL-2R alpha antibody, CD25-8D8, on the proliferative response of human peripheral blood lymphocytes. Lymphocytes from purified protein derivative (PPD)-positive donors were cultured with PPD and various concentrations of CD25-8D8 for up to 9 days, and [3H]thymidine uptake was measured. Whereas the proliferative response of human lymphocytes to PPD was suppressed by high concentrations of CD25-8D8, subinhibitory amounts of CD25-8D8 enhanced lymphocyte proliferation by 3.5-fold (range 2.2-6.2-fold) on the second day after maximal [3H]thymidine uptake had occurred. By itself, CD25-8D8 could not induce proliferation of washed 5-day PPD-activated lymphocytes during reculturing; instead, growth enhancement by CD25-8D8 was dependent on the presence of PPD-activated culture supernatant or moderate levels of exogenous IL-2. The enhancing effect of anti-IL-2R alpha antibody, observed in both murine and human systems, reinforces the possibility that binding of IL-2 to the IL-2R alpha chain plays a negative regulatory role in signal transduction.

Some (VIM12, Leu-15, 5A4.C5), but not all, Mac-1-specific monoclonal antibodies (mAb) induced a clear respiratory burst in unprimed monocytes but not in unprimed polymorphonuclear leucocytes (PMN). We showed that this monocyte stimulation occurred via formation of Mac-1 mAb-Fc gamma RI or Mac-1 mAb-Fc gamma RII complexes, as human monomeric IgG1 could completely block the respiratory burst induced by the murine IgG2a subclass anti-Mac-1 mAb Leu-15 and the Fc gamma RII-specific mAb IV.3 inhibited respiratory burst formation by IgG1 subclass anti-Mac-1 mAb VIM12 and 5A4.C5, respectively. F(ab')2 fragments of mAb VIM12 did not stimulate. This association between Mac-1 and Fc gamma RII may be due to a near spatial association between these molecules in monocytes, as we observed partial inhibition of FITC-labelled anti-Fc gamma RII mAb IV.3 binding after prior incubation with mAb VIM12. If monocytes were preincubated with mAb IV.3 or aggregated IgG, there was partial inhibition of mAb VIM12 binding. The non-stimulating anti-Mac-1 mAb (JML.H11,44, OKM1, LM2/1, Mo1) did not show any significant competition with mAb IV.3 binding to Fc gamma RII. Both non-stimulating CD18-specific mAb, however, showed strong competition with mAb IV.3 binding to Fc gamma RII. On unprimed PMN, the situation was different. No Mac-1-specific mAb induced a respiratory burst and there was no competitive inhibition between anti-Mac-1 mAb and antibodies binding to Fc gamma RII. In interferon-gamma (IFN-gamma)-primed PMN, however, we observed a functional association between Mac-1 and Fc gamma RI as IgG2a subclass mAb Leu-15 induced a respiratory burst which could be inhibited by monomeric human IgG1, as observed in monocytes. However, no other anti-Mac-1 mAb was able to induce a respiratory burst in IFN-gamma-primed PMN. Therefore, a similar signal transducing capability may exist between Mac-1 and Fc gamma RI on both monocytes and PMN, despite a different relationship between Mac-1 and Fc gamma RII on these cell populations. As no Mac-1 beta-chain-specific (CD18)mAb were able to induce a respiratory burst in monocytes, despite being able to interact with Fc gamma R via their Fc regions, as detected by competition with mAb IV.3 for binding to Fc gamma RII, we conclude that intracellular signalling via Mac-1 mAb-Fc gamma RII complexes requires the alpha-chain.