Interleukin-13 is produced by activated human CD45RA+ and CD45R0+ T cells: modulation by interleukin-4 and interleukin-12

Interleukin (IL)-13 is a cytokine originally identified as a product of activated T cells. Little is known, however, about IL-13 production by human T cells and its modulation by other cytokines. Here, we show that IL-13 is produced by activated human CD4⁺ and CD8⁺ CD45R0⁺ memory T cells and CD4⁺ and CD8⁺ CD45RA⁺ naive T cells. In contrast, IL-4, which shares many biological activities with IL-13, is only produced by CD45R0⁺ T cells following activation. Analysis of intracellular cytokine production by single CD45RA⁺ and CD45R0⁺ T cells indicated that IL-13 continued to be produced for more than 24 h after stimulation, whereas IL-4 could not be detected after 24 h. These data were confirmed by measurement of specific mRNA and suggest that IL-13, unlike IL-4, but like interferon-γ (IFN-γ), is a cytokine with long-lasting kinetics. The majority of human CD45R0⁺ T cells produced IL-4 and IL-13 simultaneously. In contrast, IFN-γ protein was generally not co-expressed with IL-4 or IL-13. IL-4 added to primary cultures of highly purified peripheral blood T cells activated by the combination of anti-CD3+anti-CD28 mAb enhanced IL-13 production by CD45RA⁺ and to a lesser extent by CD45R0⁺ T cells. Under these conditions, however, IL-12 inhibited IL-13 production by CD45RA⁺ T cells and to a lesser extent by CD45R0⁺ T cells in a dose-dependent fashion. These inhibiting effects were not related to enhanced IFN-γ production induced by IL-12, since IFN-γ by itself did not affect IL-13 production. Collectively, our data indicate that IL-13 is produced by peripheral blood T cells which also produce IL-4, but not IFN-γ, and by naive CD45RA⁺ T cells which, in contrast, fail to produce IL-4. These observations, together with the long-lasting production of IL-13, suggest that IL-13 may have IL-4-like functions in situations where T cell-derived IL-4 is still absent or where its production has already been down-regulated.     

Rapid conversion of naive to effector T cell function counteracts diminished primary human newborn T cell responses.

The reduced incidence of graft versus host disease following the use of human cord blood as a source of stem cells for bone marrow reconstitution challenges our understanding of the immunocompetence of newborn T cells. Newborn CD4+ T cells express mainly the CD45RA phenotype and have been considered to respond comparably to adult CD4+ T cells exhibiting the CD45RA phenotype. We compared the in vitro kinetics of phenotypic conversion of newborn and adult CD4+CD45RA+ T cells to CD4+CD45RO+ T cells. The cytokine profile and B cell helper activity of the converted CD4+CD45RO+ T cell population were also determined. Newborn CD4+CD45RA+ T cells were converted to CD4+CD45RO+ with significantly faster time kinetics than adult CD4+CD45RA+ T cells, following either phytohaemagglutinin (PHA) or anti-CD2 activation. Freshly purified newborn naive T cells did not produce IL-2, IL-4 or interferon-gamma (IFN-gamma) following stimulation, whereas adult naive T cells secreted IL-2 and adult-derived CD4+CD45RO+ T cells secreted all three cytokines under the same stimulatory conditions. However, newborn and adult CD4+CD45RA+ T cells, following primary stimulation and maturation in vitro, acquired the ability to secrete a Th1-type cytokine profile of IL-2 and IFN-gamma after secondary stimulation. Newborn CD4+ naive T cells that acquired the CD45RO phenotype in vitro also gained B cell helper activity equivalent to that of adult in vitro matured CD4+ naive T cells. These findings suggest that newborn and adult CD4+CD45RA+ T cell subsets are differentially responsive to various stimuli. They show that newborn CD4+CD45RA+ naive T cells can transform more quickly than their adult counterparts into functionally equivalent CD4+CD45RO+ T cells, a process that may be important to counteract the immature immune environment which exists in the newborn.     

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.     

CD28 activation promotes Th2 subset differentiation by human CD4+ cells

Ligation of CD28 provides a costimulatory signal to T cells necessary for their activation resulting in increased interleukin (IL)-2 production in vitro, but its role in IL-4 and other cytokine production and functional differentiation of T helper (Th) cells remains uncertain. We studied the pattern of cytokine production by highly purified human adult and neonatal CD4⁺ T cells activated with anti-CD3, phorbol 12-myristate 13-acetate (PMA) and ionomycin, or phytohemagglutinin (PHA) in the presence or absence of anti-CD28 in repetitive stimulation-rest cycles. Initial stimulation of CD4⁺ cells with anti-CD3 (or the mitogens PHA or PMA+ionomycin) and anti-CD28 monoclonal antibodies induced IL-4, IL-5 and interferon-γ (IFN-γ) production and augmented IL-2 production (6- to 11-fold) compared to cells stimulated with anti-CD3 or mitogen alone. The anti-CD28-induced cytokine production corresponded with augmented IL-4 and IL-5 mRNA levels suggesting increased gene expression and/or mRNA stabilization. Most striking, however, was the progressively enhanced IL-4 and IL-5 production and diminished IL-2 and IFN-γ production with repetitive consecutive cycles of CD28 stimulation. The enhanced Th2-like response correlated with an increased frequency of IL-4-secreting cells; up to 70% of the cells produced IL-4 on the third round of stimulation compared to only 5% after the first stimulation as determined by ELISPOT. CD28 activation also promoted a Th2 response in naive neonatal CD4⁺ cells, indicating that Th cells are induced to express a Th2 response rather than preferential expansion of already established Th2-type cells. This CD28-mediated response was IL-4 independent, since enhanced IL-5 production with repetitive stimulation cycles was not affected in the presence of neutralizing anti-IL-4 antibodies. These results indicate that CD28 activation may play an important role in the differentiation of the Th2 subset in humans.     

Ligation of TAPA-1 (CD81) or major histocompatibility complex class II in co-cultures of human B and T lymphocytes enhances interleukin-4 synthesis by antigen-specific CD4+ T cells

We have previously shown that CD4⁺ T cells from allergic individuals are predisposed to producing interleukin (IL)-4 in response to allergens. IL-4 production could be modulated by antigen concentration as well as by the type of antigen-presenting cells (APC), with B lymphocytes inducing greater quantities of IL-4 than monocytes. Using this system we examined IL-4 synthesis after culture of CD4⁺ T cells with B cells, monocytes, or both, as APC in the presence of allergen and a monoclonal antibody against CD81 (TAPA-1), a member of the TM4 superfamily of proteins that regulates activation, proliferation and trafficking of B cells. Addition of anti-CD81 mAb during culture enhanced IL-4 synthesis by 2- to 70-fold over that using an isotype-matched control mAb. Furthermore, anti-CD81 mAb enhanced IL-4 synthesis in CD4⁺ T cells only when CD4⁺ T cells were cultured with B cells but not monocytes as APC, indicating that anti-CD81 mAb affected IL-4 synthesis in T cells via interactions with B cells. However, pretreatment of either population separately with anti-CD81 mAb prior to culture had no effect on subsequent IL-4 synthesis, suggesting a requirement for temporal or cooperative interactions between T and B lymphocytes. In addition, anti-CD81 mAb enhanced IL-4 production but reduced CD4⁺ T cell antigen-specific proliferation, demonstrating that IL-4 production and proliferation by CD4⁺ T cells were inversely related. Finally, mAb to major histocompatibility complex class II but not to anti-CD19 also enhanced IL-4 synthesis when B lymphocytes were used as APC. In all instances, enhancement of CD4⁺ IL-4 synthesis correlated with the presence of large cell aggregates in T-B lymphocyte cocultures. These results indicate that the capacity of B cells to induce IL-4 can be significantly enhanced by ligation of particular molecules on their surface and should aid in the design of treatments for diseases in which modulation of the cytokine profile would be beneficial.     

Human CD45RA+ and CD45R0+ T cells exhibit similar CD3/T cell receptor-mediated transmembrane signaling capacities but differ in response to co-stimulatory signals

CD45RA⁺ cells have been described to be less responsive to CD3/T cell receptor (TcR)-mediated activation than CD45R0⁺ T cells. To analyze the underlying mechanism of the differential responses we compared CD3/TcR-triggered tyrosine phosphorylation in the two subsets and studied the role of co-stimulatory signals provided either by accessory cells or pharmacologic activation of protein kinase C by phorbol ester. Stimulation of purified CD45RA⁺ and CD45R0⁺ T cells with CD3/TcR antibodies induced similar patterns and intensities of tyrosine phosphorylation in the two subsets, but no proliferation. If accessory cells were used as the source of co-stimulatory signals, strong expression of the 55-kDa chain of the interleukin-2 (IL-2) receptor (CD25), significant IL-2 production and vigorous proliferation were observed in CD45R0⁺ cells, whereas CD45RA⁺ cells responded weakly. However, when CD3/TcR-mediated triggering was combined with activation of protein kinase C by phorbol ester, CD45RA⁺ cells responded strongly. These data indicate that the transmembrane signaling capacity of the T cell receptor expressed by CD45RA⁺ and CD45R0⁺ cells is similar and, therefore, is presumably not responsible for the differential reactivities of the two subsets. It is more likely that co-stimulatory signals determine whether CD3/TcR-initiated activation results in strong or weak responses.     

Cytomegalovirus infection induces the accumulation of short-lived, multifunctional CD4+CD45RA+CD27+ T cells: the potential involvement of interleukin-7 in this process

The relative roles that ageing and lifelong cytomegalovirus (CMV) infection have in shaping naive and memory CD4⁺ T-cell repertoires in healthy older people is unclear. Using multiple linear regression analysis we found that age itself is a stronger predictor than CMV seropositivity for the decrease in CD45RA⁺ CD27⁺ CD4⁺ T cells over time. In contrast, the increase in CD45RA⁻ CD27⁻ and CD45RA⁺ CD27⁻ CD4⁺ T cells is almost exclusively the result of CMV seropositivity, with age alone having no significant effect. Furthermore, the majority of the CD45RA⁻ CD27⁻ and CD45RA⁺ CD27⁻ CD4⁺ T cells in CMV-seropositive donors are specific for this virus. CD45RA⁺ CD27⁻ CD4⁺ T cells have significantly reduced CD28, interleukin-7 receptor α (IL-7Rα) and Bcl-2 expression, Akt (ser473) phosphorylation and reduced ability to survive after T-cell receptor activation compared with the other T-cell subsets in the same donors. Despite this, the CD45RA⁺ CD27⁻ subset is as multifunctional as the CD45RA⁻ CD27⁺ and CD45RA⁻ CD27⁻ CD4⁺ T-cell subsets, indicating that they are not an exhausted population. In addition, CD45RA⁺ CD27⁻ CD4⁺ T cells have cytotoxic potential as they express high levels of granzyme B and perforin. CD4⁺ memory T cells re-expressing CD45RA can be generated from the CD45RA⁻ CD27⁺ population by the addition of IL-7 and during this process these cells down-regulated expression of IL-7R and Bcl-2 and so resemble their counterparts in vivo. Finally we showed that the proportion of CD45RA⁺ CD27⁻ CD4⁺ T cells of multiple specificities was significantly higher in the bone marrow than the blood of the same individuals, suggesting that this may be a site where these cells are generated.     

Hyper-reactivity of mouse CD45RA − T cells

Mouse CD4 T cells have been partitioned into CD45RA and CD45RA^? subpopulations by means ot the monoclonal antibody 14.8. The CD45RA^? subpopulation proliferated more actively and generated more interleukin-4 (IL-4) in response to stimulation with anti-CD3 antibody and phytohemaglutinin, and more IL-2 in response to anti-CD3. This subpopulation is therefore hyper-reactive to these polyclonal stimulators, but does not show the bias towards T helper type 2 activity that has been found in studies with other related CD45 isoforms. No evidence of suppression was obtained by comparing proliferation of CD45RA^? cells in the presence and absence of CD45RA cells. Thus mouse CD4 T cells behave in these respects similarly to those of man, as is evident in a brief review of the quiescence-activation-quiescence cycle in the two species.     

CD8+ and CD45RA+ human peripheral blood lymphocytes are potent sources of macrophage inflammatory protein 1α, interleukin-8 and RANTES

The chemokines macrophage inflammatory protein 1α (MIP 1α), interleukin-8 (IL-8) and RANTES are potent regulators of leukocyte trafficking. Examination of chemokine secretion by human peripheral blood lymphocytes after stimulation with anti-CD3 or phorbol 12, 13 myristate acetate and ionomycin showed CD8⁺ cells were the dominant source of MIP 1α and RANTES. Although production of MIP 1α and IL-8 were similar in pharmacologically stimulated CD4⁺ CD45RA⁺, CD4⁺ CD45RO⁺, and CD8⁺ CD45RA⁺ cells, the largest amounts of MIP 1α and RANTES were secreted by CD8⁺ CD45RO⁺ lymphocytes. A parallel pattern of prolonged chemokine mRNA expression for at least 18 h after activation was observed in the T cell subsets. These results confirm that human T lymphocytes have a unique capacity for secretion of these three chemokines. In addition, CD8⁺ cells have an unrecognized role in recruiting cells to sites of inflammation, and adult human CD45RA⁺ cells have a physiologically significant secretory capacity.     

The CD7− T cell subset represents the majority of IL-5-secreting cells within CD4+CD45RA− T cells

Absence of CD7 is a stable phenotype in a subset of normal human T cells. Most circulating CD7⁻ T cells express the CD4⁺CD45RO⁺CD45RA⁻ memory phenotype. We analysed CD4⁺CD45RA⁻ peripheral blood lymphocytes that were separated into CD7⁺ and CD7⁻ for their in vitro cytokine secretion in response to different stimuli. The CD4⁺CD7⁻ subpopulation was found to secrete significantly higher levels of IL-5 compared with the CD4⁺CD7⁺ subset upon stimulation with ionomycin/phorbol myristate acetate (PMA) plus anti-CD28 MoAbs. In contrast to IL-5 secretion, IL-4 and interferon-gamma (IFN-γ) secretion was not significantly different in CD7⁺ and CD7⁻ T cells upon stimulation in vitro. The data indicate that the CD4⁺CD7⁻ T cell represents the majority of IL-5-secreting cells within the population of CD4⁺CD45RA⁻ memory T cells. Since CD4⁺CD7⁻ T cells were found to be enriched in various skin lesions associated with eosinophilic infiltration, the results of our study support the hypothesis that skin-infiltrating CD7⁻ T cells are one of the major sources of IL-5 responsible for the development of eosinophilic inflammation in certain skin diseases.     

Comparison of lymphokine secretion and mRNA expression in the CD45RA+ and CD45RO+ subsets of human peripheral blood CD4+ and CD8+ lymphocytes

Flow cytometric analysis of human peripheral blood T lymphocytes demonstrated that the majority of the CD4⁺ cells were CD29⁺ or CD45RO⁺ “mature” cells while the CD8⁺ cells were primarily CD45RA⁺ “naive” cells. After an initial separation into CD4⁺ and CD8⁺ cells and a secondary separation into CD45 subsets, lymphokine secretion was assessed after phorbol 12-myristate 13-acetate and ionomycin or fixed anti-CD3 stimulation. Within the respective CD45 subsets, CD4⁺ cells produced more interleukin (IL)-2, IL-4, and IL-6; but the CD8⁺ cells secreted more interferon-γ and granulocyte/macrophage-colony-stimulating factor. Tumor necrosis factor-α secretion was similar in the matched CD45 subsets. Northern analysis revealed a parallel pattern of lymphokine mRNA expression in the four lymphocyte subsets. These results suggest that human CD8⁺ peripheral blood lymphocytes have a significant capacity to secrete lymphokines, and that the low lymphokine production observed in unseparated CD8⁺ cells reflects the higher percentage of less functional CD45RA⁺ cells.     

CD3 antigen-mediated calcium signals and protein kinase C activation are higher in CD45R0+ than in CD45RA+ human T lymphocyte subsets

T lymphocytes may be separated into subsets according to their expression of CD45 isoforms. The CD45R0⁺ T cell subset has been reported to proliferate in response to recall antigen and to mitogenic mAb to a much greater extent than the CD45RA⁺ subset. This difference could be due to more efficient coupling of the T cell antigen receptor complex to mitogenic signaling pathways. To investigate this possibility, CD3 antigen-induced calcium signals, diacylglycerol (DAG) production and protein kinase C (PKC) activation levels were compared in CD45RA⁺ and CD45R0⁺ human T lymphocyte subsets derived from peripheral blood. The mean CD3-induced rise in intracellular calcium was 80% greater in CD45R0⁺ than in CD45RA⁺ cells. Basal DAG levels in CD45R0⁺ cells were found to be, on average, 60% higher than in CD45RA⁺ cells (p = 0.002), but the CD3-induced production of DAG over background was not different in the two subsets (p = 0.4). Basal PKC activity, and CD3-induced PKC activation levels over background, were found to be 50% and 140% higher, respectively, in CD45R0⁺ cells than in CD45RA⁺ cells (p = 0.015 and 0.023). The CD45R0⁺ subset contained a higher proportion of cells expressing activation markers, such as CD25, CD71 and major histocompatibility complex class II, when compared to the CD45RA⁺ subset. Our results suggest that the elevated basal DAG levels observed in the CD45R0⁺ subset may reflect the recent activation of these cells. Both the higher basal DAG and CD3-induced elevation in intracellular calcium observed in the CD45R0⁺ cells may contribute to the greater PKC activation signals triggered by CD3 mAb in this subset. These findings elucidate the greater response of CD45R0⁺ T cells to mitogenic stimuli compared to CD45RA⁺ cells.     

CD45RB expression defines two interconvertible subsets of human CD4+ T cells with memory function

Reciprocal expression of CD45RA and CD45RO in human CD4⁺ T cells defines populations understood to be naive cells (CD45RA⁺CD45RO^?) and memory cells (CD45RA^?CD45RO⁺). We investigate two subsets of CD45RA^?CD45RO⁺ CD4⁺ human T cells which differ by fourfold in their expression of the CD45RB isoform; one is CD45RB^bright and the other is CD45RB^intermediate. In contrast, CD45RA⁺ naive cells are all CD45RB^bright. Both subsets of CD45RA^? cells proliferate in response to recall antigens so we designate them MEM 1 (CD45RO⁺RB^bright) and MEM 2 (CD45RO⁺RB^intermediate). CD45RA and CD45RB expression are regulated independently during in vitro activation of naive cells. When MEM 1 cells are activated they tend to down-regulate CD45RB expression, whereas activated MEM 2 cells tend to up-regulate CD45RB expression. Thus, in contrast to the stability of the CD45RA^?CD45RO⁺ phenotype, the MEM 1 and MEM 2 phenotypes are labile and may interconvert. MEM 1 and MEM 2 cells produced comparable amounts of interleukin(IL)?2, IL?4, and IL-5 though MEM 1 cells produced slightly more interferon(IFN)-γ (mean 1.7-fold more). MEM 1 cells consistently proliferated more (mean 2.3-fold more) than MEM 2 cells early during in vitro activation. Thus, differential expression of CD45RB within CD45RA^? cells defines two subsets that have similar properties except for somewhast greater IFN-γ production and proliferative responses by MEM 1 cells. Variability in CD45RB expression may represent a mechanism for fine-tuning the responsiveness of memory cells in vivo.     

Ethanol-induced CD3 and CD2 hyporesponsiveness of peripheral blood T lymphocytes

Abstract

The functional relevance of a direct ethanol effect on the membrane structure of T lymphocytes and accessory cells (APC), as well as on signal transduction systems was studied in ten normal subjects. Ethanol incubation (80 mM for 24h) of highly purified T cells increased the number of CD4⁺/CD45RA⁺ lymphocytes. In contrast, ethanol exposure induced a drop in CD14⁺/LFA-3⁺ APC values. These changes were accompanied by faulty T-cell proliferation in response to anti-CD3 and anti-CD2 mAb and inhibition of CD3- and CD2-mediated rises in intracellular calcium and, to a lesser extent, inositol 1,4,5-triphosphate levels.

These data clearly indicate that a membrane-specific ethanol interaction both modifies surface glycoproteic and/or glycolipidic structures and alters transmembrane transduction of the activation signals.     

CD28 ligands CD80 (B7-1) and CD86 (B7-2) induce long-term autocrine growth of CD4+ T cells and induce similar patterns of cytokine secretion in vitro

The interaction of CD28 and its ligands is critical for antigen-inducedT cell activation. Recent studies have demonstrated the existenceof at least two members of the B7 receptor family. In this report,the co-stimulatory signals provided by CD80 (B7-1) or CD86 (B7-2)were compared to CD28 ligation by mAb. We demonstrate that thekinetics of induction of T cell proliferation after anti-CD3stimulation was similar regardless of the form of co-stimulation.Similarly, B7-1 and B7-2 could both maintain long-term expansionof CD4 cells. The co-stimulatory effects of both B7-1 and B7-2were dependent on CD28 cross-linking, based on complete inhibitionof proliferation by CD28 antibody Fab fragments. Co-stimulationwith B7-1 and B7-2 induced high levels of cytokine secretionby resting T cells, and the effects of B7-1 and B7-2 could notbe distinguished. This conclusion is based on analysis of theinitial activation of CD28⁺ T cells. as well as T cell subpopulationsconsisting of CD4⁺ and CD8⁺ T cells. Both B7-1 and B7-2 couldelicit IL-4 secretion from CD4⁺ T cells while anti-CD28 antibodyinduced substantially less IL-4 secretion. Furthermore, bothB7-1 and B7-2 could stimulate high levels of IFN- and IL-4 fromCD4⁺CD45RO⁺ cells, while neither B7 receptor could co-stimulateIFN- and IL-4 secretion from CD4⁺CD45RA⁺ T cells. B7-1 and B7-2could, however, co-stimulate CD4⁺CD45RA⁺ T cells to secreteIL-2. By contrast, when previously activated T cells were tested,re-stimulation of CD4⁺ T cell blasts with B7-1 or B7-2 resultedin higher secretion of IL-4 and IL-5 than anti-CD28, while re-stimulationwith anti-CD28 antibody maintained a higher level of secretionof IL-2 and IFN- than B7-1 or B7-2. These observations may haveimportant implications because they suggest that the mannerof CD28 ligation can be a critical determinant in the developmentof cytokine secretion that corresponds to T_h1- and T_h2-likepatterns of differentiation. Together these observations suggestthat there are no Intrinsic differences between B7-1 and B7-2in their ability to co-stimulate the populations of cells thatwe have tested.     

Anti-CD5 extends the proliferative response of human CD5+ B cells activated with anti-IgM and interleukin-2

The CD5 T cell glycoprotein which is expressed by a subset of B cells has been shown to be involved in T cell activation and proliferation. No similar studies, to date, have addressed the role of CD5 on the B cell subset. CD5⁺ and CD5^? B cells were sorted and stimulated with anti-CD5 monoclonal antibody (mAb) in vitro. The activation and proliferative responses of these two populations, as measured by analysis of proliferation marker, did not differ following anti-μ and interleukin (IL)-2 stimulation. The addition of anti-CD5 did not change the responsiveness of such activated CD5⁺ B cells but resulted in a decrease in CD25 expression. Pre-activation of B cells with phorbol 12-myristate 13-acetate, which increased CD5 expression, failed to alter the proliferative response of CD5⁺ B cells to anti-μ and IL-2 with or without addition of anti-CD5 mAb. Anti-μ and IL-2 treatment of CD5⁺ cells resulted in optimal proliferation measured at day 3 which decreased by day 6. However, addition of anti-CD5 mAb at day 3 prevented this decline in proliferative response. This dose-dependent effect was observed only when the anti-CD5 mAb was presented to the B cells in cross-linked form. Co-stimulation of CD5 did not lower the threshold of antigen to which the B cells responded. Taken together, these data support a functional role for CD5 on B cells acting as an accessory signal, following their primary activation through the B cell receptor complex and highlight differences in the role of CD5 associated with the T cell receptor complex.     

Regulation of 4-1BB expression by cell-cell interactions and the cytokines,interleukin-2 and interleukin-4

4-1BB expression increased gradually following T cell activation, and by day 3 post-stimulation with immobilized anti-CD3 (anti-CD3i) or concanavalin A (Con A), splenic T cells were routinely 35–45% 4-1BB⁺ by flow cytometric analysis. 4-1BB was expressed on activated CD8⁺, CD4⁺, CD28⁺ and CD45RB⁺ T cells. Optimal 4-1BB expression was seen by day 6 post-stimulation and was cell density dependent. When T cells were cultured for 6 days at 1 × 10⁶/well in a 24-well plate with anti-CD3i, 82% of the cells were 4-1BB⁺. In contrast, at lower cell densities (4 × 10⁵, 2 × 10⁵ and 1 × 10⁵), optimal 4-1BB expression was observed only if the cultures were supplemented with recombinant interleukin-2 (IL-2) or recombinant IL-4 (IL-4). In agreement, with these results, modes of inducing endogenous IL-2 production such as cross-linking the costimulatory molecule, CD28, or the addition of syngeneic accessory cells to T cells activated with anti-CD3i, resulted in high levels of 4-1BB expression. The addition of interleukin-1α(IL-1α) or interferon-γ (IFN-γ) did not increase 4-1BB expression on anti-CD3i-activated T cells. In addition, if T cells were incubated with IL-2, IL-4, IL-1α, IFN-γ or anti-CD28 alone, no 4-1BB expression was induced. T cells activated with soluble anti-CD3 (anti-CD3s) in the presence of IL-2, IL-4, or accessory cells, did not express higher levels of 4-1BB on their cell surface. These data suggest that initial events crucial for efficient T cell activation, such as signals delivered through the T cell receptor/CD3 complex and the CD28 molecule, are instrumental in regulating subsequent 4-1BB expression.     

Deficient interleukin-10 production by neonatal T cells does not explain their ineffectiveness at promoting neonatal B cell differentiation

Neonatal T cells are poor promoters of Ig secretion by neonatal B cells. Since IL-10 has been shown to play a role in B cell differentiation, we investigated the relationship of IL-10 production by neonatal T cells and their ability to provide B cell help. Neonatal CD4⁺(CD8⁻) T cells and adult naive CD4⁺ (CD8⁻ / CD45RO⁻) T cells activated with immobilized anti-CD3 produced consistently less IL-10 than adult memory CD4⁺(CD8⁻ / CD45RA⁻) T cells. Production of IL-10 by adult and neonatal T cells was dependent on IL-2, but was unaffected by supplemental IL-4. Despite diminished IL-10 production, supplemental IL-10 increased neonatal T cell-dependent Ig secretion only modestly, but did not increase Ig heavy chain isotype switching. This contrasted with the ability of IL-10 to enhance the secretion of all Ig isotypes by adult B cells stimulated in the presence of either IL-2 or IL-4. These results suggest that IL-10 can promote T cell-dependent Ig secretion but not Ig heavy chain isotype switching by neonatal B cells. However, deficient IL-10 production alone does not account for the poor ability of neonatal T cells to support neonatal B cell Ig production.     

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.