Treatment with an anti-CD4 monoclonal antibody strongly ameliorates established rat adjuvant arthritis

Some experimental arthritic diseases can be prevented by treatment with anti-CD4 MoAbs. Trials with ongoing disease have not been successful so far. The aim of this study was to ascertain whether W3/25 could reverse adjuvant arthritis (AA), when beginning treatment on day 14, i.e. when the disease was established. Moreover, one group of animals treated with the anti-CD4 MoAb received OX8 MoAb at the same time, thus depleting CD8⁺ cells from circulation. During treatment with W3/25, a strong amelioration of inflammatory signals was observed, as assessed by means of paw volume increase and arthritic score. However, when treatment stopped, a rebound to arthritis signals occurred. The parallel depletion of CD8⁺ cells did not modify these effects, thus the combined treatment W3/25+OX8 gave the same amelioration as treatment with W3/25 alone. These findings indicate that CD4⁺ cells play an important role in perpetuating rat AA. Moreover, CD8⁺ cells do not seem to have a regulatory role in the CD4⁺ cells responsible for the inflammatory response.     

Rapid re-expression of CD45RC on rat CD4 T cells in vitro correlates with a change in function

Rat CD4⁺ T cells are divided phenotypically by the anti-CD45RC monoclonal antibody OX22 into subsets with contrasting functions. Stimulation of T cells in vitro is known to induce a change in isoform from CD45RC⁺ to CD45RC^?. We have investigated the in vitro conditions which promote a switch in isoform in the opposite direction. We observed that a majority of CD45RC^? CD4 T cells (> 90%) spontaneously re-expressed CD45RC during the first 1-3 days of culture in both the presence and absence of alloantigen. The T cells remained CD45RC⁺ when cultured for 7 days in serum-free growth medium. However, alloantigen-activated lymphocytes, expressing the interleukin-2 receptor (IL-2R), down-regulated CD45RC by day 4 and remained CD45RC^? during the course of the experiment. Using mixtures of allotype-marked CD45RC⁺ and CD45RC^?T cells, it was demonstrated that each subset showed comparable survival, IL-2R expression and time courses of activation in response to alloantigen. The repertoire of neither subset was, therefore, deficient in terms of allorecognition. The rapid re-expression of CD45RC in culture was accompanied by a change in function: CD45RC⁺ “converts”, obtained by overnight culture of CD45RC^? T cells, induced significantly higher graft-versus-host responses. Thus, the transition in culture from CD45RC^? to CD45RC⁺ reflects a major functional reprogramming of the cell and not a trivial modulation of a surface antigen.     

Mechanisms of T cell-induced glomerular injury in anti-glomeruler basement membrane (GBM) glomerulonephritis in rats

The effector mechanisms of T cell-dependent acute glomerular injury were studied in autologous phase anti-GBM glomerulonephritis (GN) in rats. Acute proliferative GN was induced in sensitized rats by a subnephritogenic dose of sheep anti-rat GBM antibody. Injury was manifested by proteinuria and glomerular leucocyte infiltration composed predominantly of macrophages but also CD4⁺ and CD8⁺ T cells. T cell depletion, using an anti-CD5 MoAb, demonstrated that glomerular leucocyte infiltration and proteinuria were T cell-dependent. Inhibition of T helper cell function using an anti-CD4 MoAb prevented proteinuria and glomerular macrophage and CD4⁺ T cell influx, but not accumulation of CD8⁺ T cells. Depletion of CD8⁺ T cells also prevented proteinuria and the influx of macrophages and CD8⁺ T cells, but not accumulation of CD4⁺ T cells. Macrophage depletion, using micro-encapsulated clodronate, prevented proteinuria and glomerular macrophage infiltration, but not the accumulation of CD4⁺ or CD8⁺ T cells, indicating that macrophages are the common cellular effectors for both CD4 and CD8 T cell-dependent injury. Evidence for cytotoxic mechanisms of injury (increased numbers of apoptotic cells or accumulation of natural killer (NK) cells in glomeruli) could not be demonstrated. These data suggest that acute glomerular injury in anti-GBM GN is the result of macrophage recruitment, which is dependent on both CD4 and CD8 T cells, and that direct T cell-mediated injury (cellular cytotoxicity) is not involved.     

Membrane CD45R isoform exchange on CD4 T cells is rapid,frequent and dynamic in vivo

CD4 T cells bearing high (240–190 kDa) and low (180 kDa) molecular mass isoforms of the leukocyte common antigen CD45 define functionally distinct subsets which have been equated with naive and memory T cells. In the rat, CD4 T cells expressing a high molecular mass isoform [identified by monoclonal antibody MRC-OX22 (anti-CD45RC)] exchange this for the 180 kDa molecule (CD45RC^?) when stimulated by antigen. Here we show, by transferring mature allotype-marked CD45RC^? CD4 T cells (depleted of immature Thy-1⁺ CD45RC^? recent thymic emigrants) into normal euthymic recipients, that many T cells re-express the high molecular mass isoform in less than 6 h. By 24 h, 30–60% of CD45RC^? CD4 T cells became CD45RC⁺; within a week the entire cohort appeared to exchange the low for the high molecular mass isoform. Isoform exchange was dynamic and many CD4 T cells returned once again to the CD45RC^? state. CD45RC^? CD4 T cells declined in number more rapidly than the CD45RC⁺ subset after transfer. The results suggest that CD45R isoforms distinguish between resting T cells (CD45RC⁺) and those which have encountered antigen in the recent past. CD45R isoforms would appear to be unsuitable markers of naive and memory T cells.     

CD8+ T cells and not CD4+ T cells are hyporesponsive to CD28- and CD40L-mediated activation in HIV-infected subjects

T cell dysfunction in HIV-infected subjects could be the consequence of altered sensitivity of CD4⁺ or CD8⁺ T cells to various costimulatory signals. Therefore, we studied proliferation and cytokine production in highly purified CD8⁺ and CD4⁺ T cells from HIV-infected and HIV⁻ subjects, induced by co-activation via cell-bound CD80, CD86 and CD40 or by allo-activation. Regardless of the nature of the first and the costimulatory signal, CD8⁺ T cells from patients proliferated consistently less than controls, while responses from CD4⁺ T cells were similar in patients and controls. This phenomenon was observed after ligation of CD28 combined with anti-CD3 or phorbol myristate acetate (PMA), but also after allogeneic stimulation and after activation by CD40 and anti-CD3. Anti-CD3 combined with CD80 or CD86 induced a mixed Th1/Th2-type cytokine profile in both CD4⁺ and CD8⁺ T cells from controls, whereas anti-CD3 plus CD40 induced only low levels of Th2-type cytokines and no interferon-gamma (IFN-γ) in CD4⁺ T cells. Compared with controls, CD4⁺ T cells from patients produced slightly lower levels of IL-10 but equal amounts of IFN-γ, IL-4 and IL-5, while CD8⁺ T cells from patients produced less of all cytokines tested. In conclusion, responses of purified CD4⁺ T cells from HIV⁺ subjects to various costimulatory pathways are relatively intact, whereas CD8⁺ T cells are hyporesponsive at the level of proliferation and cytokine production. A generalized intrinsic CD8⁺ T cell failure might contribute to viral and neoplastic complications of HIV infection.     

Low T cell reactivity to combined CD3 plus CD28 stimulation is predictive for progression to AIDS: correlation with decreased CD28 expression

In 219 HIV-1-infected men of the Amsterdam cohort we measured CD4⁺ T cell numbers and in vitro T cell responses to CD3 MoAbs with or without CD28 costimulation and phytohaemagglutinin (PHA). The value of these markers was estimated for disease progression within 4 years. CD28 expression on T cells has been related to T cell responses. CD28 costimulation considerably enhanced T cell reactivity (≈8–10-fold) with lower coefficients of variation compared with reactivity to CD3 MoAb alone (median 5 versus 20). T cell reactivity to CD3 plus CD28 MoAb was decreased during HIV-1 infection and was besides CD4⁺ T cell numbers the only independent predictor for progression to AIDS. Compared with the group with high CD4⁺ T cell numbers the relative risk (RR) for the group with intermediate levels was 2.28, with low levels 5.20. In the groups with intermediate and low CD3 plus CD28 responses the RR was 2.04 and 4.16, respectively. The combined RR for both was 4.65 and 21.63. The independence of this marker was confirmed when the group with low CD4⁺ T cell numbers was subdivided into groups with high, intermediate and low T cell responses. The expansion of CD8⁺CD28⁻ T cells was already apparent in HIV⁻ homosexual men, but CD8⁺CD28⁺ T cells specifically decreased in patients with AIDS. CD28 expression on T cells correlated moderately with T cell responses to CD3 plus CD28 MoAb. T cell reactivity to CD3 MoAb in the presence of CD28 MoAb is a stronger prognostic marker than T cell reactivity to CD3 MoAb alone.     

Abnormal CD45RC expression and elevated CD45 protein tyrosine phosphatase activity in LEC rat peripheral CD4+ T cells

LEG rats are known to show a maturational arrest in the development of CD4⁺8⁺ to CD4⁺8^? cells in the thymus. Despite the blockade of maturation of CD4⁺8^? thymocytes, CD4⁺ T cells were observed in peripheral lymphoid organs, and these cells exhibit a defect in interleukin-2 (IL-2) production upon concanavalin A (Con A) stimulation. Although peripheral CD4⁺ cells in normal rat highly expressed CD45RC (CD45RC^high), the level of CD45RC expression was low (CD45RC^low) in LEC rat peripheral CD4⁺ cells. However, CD4⁺ cells from both strains highly expressed CD45 when those cells were stained by pan-CD45 mAb, suggesting that LEC rat CD4⁺ cells are deficient in expression of the CD45RC isoform, but not of CD45 molecules. When backcross rats from (F344 × LEC)F₁ × LEC were examined, the phenotype for CD45 expression pattern in CD4⁺ cells was clearly correlated with IL-2 production level in response to Con A stimulation. Thus, CD45RC^low cells exhibit a defect in IL-2 production, while CD45RC^high cells show normal IL-2 production. Protein tyrosine phosphatase (PTPase) activity in the membrane fraction of LEC rat CD4⁺ cells was threefold higher than that of normal rat CD4⁺ cells. Con A stimulation led to an increase in tyrosine phosphorylation levels, especially 100- and 40-kDa proteins, in normal rat CD4⁺ cells. In LEC rat CD4⁺ cells, however, the level of tyrosine phosphorylation in those proteins were very low. These results suggest that an elevated CD45 PTPase activity is responsive for a defect in IL-2 production in LEC rat peripheral CD4⁺ T cells.     

Synergistic OX40 and CD30 signals sustain CD8+ T cells during antigenic challenge

Prior to acquiring a memory phenotype, antigen‐activated CD8⁺ T cells need to expand and then undergo a contraction phase. Utilizing two different antigenic stimuli, we provide evidence that the tumor necrosis factor receptors OX40 and CD30 integrate synergistic signals during the expansion phase to help maintain CD8⁺ effectors. Thus, double deficiency in OX40 and CD30 leads to CD8⁺ cell loss during expansion after immunization either with OVA or with murine CMV. Following their contraction, OX40‐ and CD30‐deficient CD8⁺ T cells persist normally in CMV‐infected mice. In contrast, persistence after OVA challenge is dependent on OX40 and CD30. Collectively, our data define the important role of both OX40 and CD30 during CD8⁺ T‐cell activation, and show that long‐term CD8 persistence after contraction is regulated not only by stimulatory receptors but also by the nature of the antigen or how the antigen is presented.     

Distinct roles for CD4 and CD8 as co-receptors in T cell receptor signalling

We demonstrate that CD4 and CD8 modify signals induced through the T cell receptor for antigen (TCRαβ) in distinct fashions. Pretreatment of CD4⁺ lymph node T cells with CD4-specific monoclonal antibody results in a tenfold inhibition of DNA synthesis induced by anti-TCRαβ. In contrast, pretreatment of CD8⁺ T cells with CD8-specific mAb has no effect on DNA synthesis subsequently induced through TCRαβ. While inhibiting late activation signals, pretreatment with anti-CD4 does not detectably alter the pattern of anti-TCRαβ-induced tyrosine phosphorylation of cellular proteins, nor subsequent Ca²⁺ mobilization. The distinct biological consequences of anti-CD4 and anti-CD8 pretreatment correlate with the differential association of their respective ligands with the cellular protein tyrosine kinase, p56^lck. While both T cell lineages contain similar levels of cellular p56^lck, tenfold more is associated with CD4 than with CD8. This difference is associated with the differential effects of pretreatment with anti-CD4 and anti-CD8 on the distribution and activity of p56^lck. Further, antibody-mediated aggregation of TCRαβ on CD4⁺ T cells induces the appearance of a p56^lck species with decreased mobility in sodium dodecylsulfate-polyacrylamide gel electrophoresis. This effect is observed in CD4⁺ T cells exclusively and involves the fraction of p56^lck which is not associated with CD4. The results presented here demonstrate that the signalling elements which couple the antigen receptor to second messenger-generating systems are under distinct physical and/or functional constraints in the two T cell lineages.     

Distinct involvement of CD45 in antigen receptor signalling in CD4+ and CD8+ primary T cells

We demonstrate that pretreatment of primary CD4⁺, but not CD8⁺ T cells with anti-CD45 inhibits activation signals induced through the T cell receptor for antigen (TCRαβ). Specifically, anti-TCRαβ-mediated tyrosine phosphorylation of phospholipase C-γ1 is inhibited, and this in turn correlates with the inhibition of subsequent Ca²⁺ mobilization and DNA synthesis. In marked contrast, none of these activation parameters are affected by anti-CD45 in CD8⁺ T cells. Perturbation of TCRαβ signalling in CD4⁺ cells is observed in conditions which do not detectably affect the level of CD45 expression, or its membrane distribution. Further, changes in the intrinsic phosphatase activity of CD45 are not detectable. While anti-CD45 ablates TCRαβ signalling, anti-CD3?-mediated activation is unaffected. This suggests that elements of the antigen receptor complex can be functionally uncoupled, and indicates that the requirements for CD45 in signalling through these two elements are different. The results demonstrate that the involvement of CD45 in coupling TCRαβ to second messenger-generating pathways is under distinct physical and/or functional constraints in primary CD4⁺ and CD8⁺ T cells.     

Anomalies of the CD8+ T cell pool in haemochromatosis: HLA-A3-linked expansions of CD8+CD28− T cells

The present study consists of a phenotypic and functional characterization of peripheral blood T lymphocytes in a group of 21 patients with hereditary haemochromatosis (HH), an MHC class I-linked genetic disease resulting in iron overload, and a group of 30 healthy individuals, both HLA-phenotyped. The HH patients studied showed an increased percentage of CD8⁺ CD28⁻ T cells with a corresponding reduction in the percentage of CD8⁺ CD28⁺ T cells in peripheral blood relative to healthy blood donors. No anomalies of CD28 expression were found in the CD4⁺ subset. The presence of the HLA-A3 antigen but not age accounted for these imbalances. Thus, an apparent failure of the CD8⁺ CD28⁺ T cell population ‘to expand’, coinciding with an ‘expansion’ of CD8⁺ CD28⁻ T cells in peripheral blood of HLA-A3⁺ but not HLA-A3⁻ HH patients was observed when compared with the respective HLA-A3-matched control group. A significantly higher percentage of HLA-DR⁺ but not CD45RO⁺ cells was also found within the peripheral CD8⁺ T cell subset in HH patients relative to controls. Phytohaemagglutinin (PHA) stimulation of peripheral blood mononuclear cells (PBMC) for 5 days showed: (i) that CD8⁺ CD28⁺ T cells both in controls and HH were able to expand in vitro; (ii) that CD8⁺ CD28⁻ T cells decreased markedly after activation in controls but not in HH patients. Moreover, functional studies showed that CD8⁺ cytotoxic T lymphocytes (CTL) from HH patients exhibited a diminished cytotoxic activity (approx. two-fold) in standard ⁵¹Cr-release assays when compared with CD8⁺ CTL from healthy controls. The present results provide additional evidence for the existence of phenotypic and functional anomalies of the peripheral CD8⁺ T cell pool that may underlie the clinical heterogeneity of this iron overload disease. They are of particular relevance given the recent discovery of a novel mutated MHC class I-like gene in HH.     

Costimulation of CD3/TcR complex with either integrin or nonintegrin ligands protects CD4+ allergen-specific T-cell clones from programmed cell death

An optimal stimulation of CD4⁺ cells in an immune response requires not only signals transduced via the TcR/CD3 complex, but also costimulatory signals delivered as a consequence of interactions between T-cell surface-associated costimulatory receptors and their counterparts on antigen-presenting cells ‘APC). The intercellular adhesion molecule-1 ‘ICAM-1, CD54) efficiently costimulates proliferation of resting, but not antigen-specific, T cells. In contrast, CD28 and CD2 support interleukin ‘IL)-2 synthesis and proliferation of antigen-specific T cells more efficiently than those of resting T cells. The molecular basis for this differential costimulation of T cells is poorly understood. Cypress-specific T-cell clones ‘TCC) were generated from four allergic subjects during in vivo seasonal exposure to the allergen. Purified cypress extract was produced directly from fresh collected pollen and incubated with the patients' mononuclear cells. Repeated allergen stimulation was performed in T-cell cultures supplemented with purified extract and autologous APC. The limiting-dilution technique was then adopted to generate allergen-specific TCC, which were also characterized by their cytokine secretion pattern as ThO ‘IL-4 plus interferon-gamma) or Th2 ‘IL-4). Costimulation-induced proliferation or apoptosis was measured by propidium iodide cytofluorometric assay. By cross-linking cypress-specific CD4⁺ and CD8⁺ T-cell clones with either anti-CD3 or anti-CD2, anti-CD28, and anti-CD54 monoclonal antibodies, we demonstrated that CD4+ clones ‘with ThO- or Th2-type cytokine production pattern) undergo programmed cell death only after anti-CD3 stimulation, whereas costimulation with either anti-CD54 or anti-CD28 protects target cells from apoptosis. The costimulation-induced protection from apoptotic death was associated with a significant rise in IL-4 secretion in both Th0 and Th2-type clones. In contrast, cypress-specific Th0 CD8⁺ clones were more susceptible to stimulation-induced apoptosis via either anti-CD3 or anti-CD2, alone or in combination with anti-CD54 or anti-CD28, thus displaying only slight but nonsignificant modifications in the pattern of IL-4 secretion. The death-promoting costimulatory effects were not observed with highly purified normal resting CD4⁺ or CD8⁺ lymphocytes. Taken together, these results suggest that TcR engagement by an allergen in the context of functionally active APC induces activation-dependent cell death of some, perhaps less specific, cells, and this may be an important homeostatic mechanism through which functional expansion of allergen-specific T cells is regulated during an ongoing immune response.     

Antigen-independent adhesion of CD4 CD45RA T cells from cord blood

Antigen-independent adhesion of resting adult CD4⁺ CD45RO⁺ T cells to B lymphocytes has been shown to be transient and can be down-regulated by CD4 major histocompatibility complex (MHC) class II molecule interactions. Conversely, adhesion of adult CD4+ CD45RA+ subpopulation to B cells is not regulated by ligands of CD4. We have investigated the regulation of adhesion of cord blood CD45RA⁺ CD4⁺ T lymphocytes. In contrast to adult CD45RA⁺ CD4⁺ T cells, cord blood CD45RA⁺ CD4⁺ T cells were strongly sensitive to the down-regulation of adhesion mediated by the CD4-HLA class II interaction, since adhesion to MHC class II(⁺) B cells was transient and inhibited by an anti-CD4 antibody. In addition, human immunodeficiency virus gpl60, synthetic gpl06-derived peptides encompassing a CD4 binding site inhibited conjugate formation between cord blood CD45RA⁺ CD4+ T cells and B cells. Following activation of the cord blood CD4 T cells by an anti-CD3 antibody, a conversion from a transient to a stable adhesion pattern of cord blood CD4 T cells to B cells occurred in 2 days. The reversal to a transient adhesion occurred at day 8 following anti-CD3 activation in correlation with a complete shift to a CD45RO phenotype of the cord blood CD4 T cells. These data suggest that CD4 T cell adhesion can be developmentally regulated.     

The NH2-terminal domain of rat CD2 binds rat CD48 with a low affinity and binding does not require glycosylation of CD2

CD2, CD48 and CD58 are structurally similar cell adhesion-molecules forming a subset of the immunoglobulin superfamily (IgSF). In humans CD58 is a ligand for CD2 while in mice CD2 binds CD48. We constructed a soluble chimeric molecule comprising the extracellular portion of rat CD48 and domains 3 and 4 of rat CD4 (sCD48-CD4) and used it to examine whether CD2 is a ligand for CD48 in rats. sCD48-CD4-coated polystyrene Dynabeads? formed rosettes on rat CD2-transfected COS-7 cells, and this rosetting was blocked by anti-CD2 (OX34) and anti-CD48 (OX45) monoclonal antibodies. We used sucrose-gradient ultracentri-fugation to show that sCD48-CD4 binds, in solution, to soluble forms of rat CD2 including the single NH₂-terminal IgSF domain of rat CD2 expressed in bacteria. The upper limit of the affinity of the rat CD48-CD2 interaction is 4 × 10⁵ M^?1, lower than the published affinity of human CD2 for CD58. These results show that rat CD48 binds CD2 on its NH₂-terminal IgSF domain with a low affinity and that binding is independent of glycosylation.     

Major histocompatibility complex class I-restricted CD8+ T cells and class II-restricted CD4+ T cells,respectively, mediate and regulate contact sensitivity to dinitrofluorobenzene

Contact sensitivity (CS) is a form of delayed-type hypersensitivity to haptens applied epicutaneously and is thought to be mediated, like classical delayed-type hypersensitivity responses, by CD4⁺ T helper-1 cells. The aim of this study was to identify the effector T cells involved in CS. We studied CS to the strongly sensitizing hapten dinitrofluorobenzene (DNFB) in mice rendered deficient by homologous recombination in either major histocompatibility complex (MHC) class I, MHC class II, or both, and which exhibited deficiencies in, respectively, CD8⁺, CD4⁺, or both, T cells. MHC class I single-deficient and MHC class I/class II double-deficient mice, both of which have a drastic reduction in the number of CD8⁺ T cells, were unable to mount a CS response to DNFB. In contrast, both MHC class II-deficient mice and normal mice treated with an anti-CD4 monoclonal antibody (mAb) developed exaggerated and persistent responses relative to heterozygous control littermates. Furthermore, anti-CD8 mAb depletion of class II-deficient mice totally abolished their ability to mount an inflammatory response to DNFB. Removal of residual CD4⁺ T cells in class II-deficient mice by anti-CD4 mAb treatment did not diminish the intensity of CS. These data clearly demonstrate that class I-restricted CD8⁺ T cells are sufficient for the induction of CS to DNFB, and further support the idea that MHC class II-restricted CD4⁺ T cells down-regulate this inflammatory response.     

Lysis of CD4+ lymphocytes by non-HLA-restricted cytotoxic T lymphoe from HIV-infected individuals

Individuals infected with HIV have elevated numbers of total and activated CD8⁺ lymphocytes in peripheral blood. CD8⁺ lymphocytes from HIV-infected individuals have been shown to mediate non-human histocompatibility-linked antigen (HLA)-restricted suppression of viral replication, HLA-restricted killing of cells expressing HIV antigens, and killing of uninfected lymphocytes. We studied CD8⁽ T lymphocytes that lysed autologous CD4+ lymphocytes, hetcrologous CD4¹ lymphocytes from HIV-infected individuals and uninfected CD4⁺ lymphocytes. Killing in all cases required T cell receptor (TCR)-mediated recognition or triggering. However, these CD8 cytotoxic T lymphocytes (CTL) killed HLA class I mismatched CD4* lymphocytes and CD4⁴ lymphocytes treated with a MoAb against HLA-A, B and C antigens (PA2.6) which blocks HLA class I-restricted killing. HLA class H-negativc CD4* T lymphoma cells (CEM.NK^R) were also killed by anti-CD3 inhibited CTL. Stimulation of peripheral blood lymphocytes (PBL) from HIV-infected individuals, but not uninfected controls, with concanavalin A (Con A) and IL-2, induced non-HLA-restricted TCR aft¹, CD8^f CTL which lysed CD4⁺ lymphocytes. Activation ofCD4’lymphocytes increased their susceptibility to CD8^f CTL-mediated lysis. In HIV infection, a population of non-HLA-restricted CTL which lyse activated CD4⁺ lymphocytes is expanded. The expansion of CTL with unusual characteristics is interesting, because the stimulus for this expansion is unknown. CTL which recognize activated CD4⁺ cells could play a role in immune regulation and the pathogenesis of A IDS.     

CD40 ligand-positive CD8+ T cell clones allow B cell growth and differentiation

A fraction of activated CD8⁺ T cells expresses CD40 ligand (CD40L), a molecule that plays a key role in T cell-dependent B cell stimulation. CD8⁺ T cell clones were examined for CD40L expression and for their capacity to allow the growth and differentiation of B cells, upon activation with immobilized anti-CD3. According to CD40L expression, CD8⁺ clones could be grouped into three subsets. CD8⁺ T cell clones expressing high levels of CD40L (≥80% CD40L⁺ cells) were equivalent to CD4⁺ T cell clones with regard to induction of tonsil B cell proliferation and immunoglobulin (Ig) production, provided the combination of interleukin (IL)-2 and IL-10 was added to cultures. CD8⁺ T cell clones, with intermediate levels of CD40L expression (10 to 30% CD40L⁺ cells), also stimulated B cell proliferation and Ig secretion with IL-2 and IL-10. B cell responses induced by these CD8⁺ T cell clones were neutralized by blocking monoclonal antibodies specific for either CD40L or CD40. By contrast, CD40L^?? T cell clones (?5 % CD40L⁺ cells), only induced marginal B cell responses even with IL-2 and IL-10. All three clone types were able to activate B cells as shown by up-regulation of CD25, CD80 and CD86 expression. A neutralizing anti-CD40L antibody indicated that T cell-dependent B cell activation was only partly dependent on CD40-CD40L interaction. These CD40L^?? clones had no inhibitory effects on B cell proliferation induced by CD40L-expressing CD8⁺ T cell clones. Taken together, these results indicate that CD8⁺ T cells can induce B cell growth and differentiation in a CD40L-CD40-dependent fashion.     

Role of the CD40-CD40 ligand interaction in CD4+ T cell contact-dependent activation of monocyte interleukin-1 synthesis

Most studies of the induction of cytokine synthesis in monocytes have employed an exogenous triggering agent such as lipopolysaccharide. However, in nonseptic inflammatory responses (e.g. rheumatoid arthritis) monocyte activation occurs as a result of T cell-generated signals. In previous reports, we and others have demonstrated that contact-dependent T cell-generated signals are capable of contributing to macrophage activation. We have shown that plasma membranes from anti-CD3 activated purified peripheral CD4⁺ T cells (Tm^A) but not from resting CD4⁺ cells (Tm^R) induce monocytes to synthesize interleukin (IL)-1 in the absence of co-stimulatory cytokines. Studies to determine the expression kinetics of the molecule(s) unique to activated CD4⁺ T cells which interact with monocytes to induce IL-1 revealed that optimal expression occurred at 6 h post activation. This matched the previously reported kinetics of expression of CD40 ligand (CD40L) on activated peripheral T cells, implicating the CD40-CD40L interaction as a candidate for the initiator of the IL-1 signaling event. The ability of Tm^A to induce IL-1 synthesis in resting monocytes could be markedly reduced by addition of a monoclonal anti-CD40L antibody, 5c8. In addition, a monoclonal anti-CD40 IgM (BL-C4) proved dramatic in its ability to induce resting monocytes to synthesize IL-1. In summary, these results demonstrate that the CD40-CD40L interaction provides a critical component of CD4⁺ T cell contact-dependent activation of monocyte IL-1 synthesis.     

Defective integration of activating signals derived from the T cell receptor (TCR) and costimulatory molecules in both CD4+ and CD8+ T lymphocytes of common variable immunodeficiency (CVID) patients

CVID is characterized by hypogammaglobulinaemia and impaired antibody production. Previous studies demonstrated defects at the T cell level. In the present study the response of purified CD4⁺ and CD8⁺ T lymphocytes to stimulation with anti-TCR monoclonal antibody (the first signal) in combination with anti-CD4 or anti-CD8, anti-CD2 and anti-CD28 MoAbs (the costimulatory signals) was investigated. Both CD4⁺ and CD8⁺ T cells from the patients showed significantly reduced IL-2 release following stimulation via TCR and costimulation via CD4 or CD8 and CD2, respectively. However, normal IL-2 production following TCR plus phorbol myristate acetate (PMA) costimulation and normal expression of an early activation marker, CD69, after TCR + CD28 stimulation indicated that TCR was able to transduce a signal. Furthermore, both IL-2 and IL-4 release were impaired in CD4⁺ lymphocytes following TCR + CD28 stimulation. In addition, stimulation via TCR + CD28 resulted in significantly decreased expression of CD40 ligand in the patients. These results suggest that the integration of activating signals derived from the TCR and costimulatory molecules is defective in CVID patients; the defect is not confined to costimulation via a single molecule, or restricted to cells producing Thl-type cytokines such as IL-2, and is expressed in both CD4⁺ and CD8⁺T cell subsets.