Crosslinking of the CD5 antigen on human T cells induces functional IL2 receptors.

CD5 is a 67-kDa antigen that is expressed on the membrane of the majority of human T cells, and on a subset of B cells. Previous studies have demonstrated that anti-CD5 monoclonal antibodies (mAb) can provide a helper signal for T cell activation through the TCR/CD3 complex. We now demonstrate that when CD5 is crosslinked by immobilized anti-CD5 mAb in the absence of other activating stimuli, the T cells proliferate in response to recombinant interleukin 2 (rIL2) (but not to rIL4). Four different anti-CD5 mAb (anti-Leu1, 10.2, anti-T1, and OKT1) had a similar effect. IL2 responsiveness could be induced with immobilized anti-CD5 mAb in cultures of purified T cells, but was enhanced by the addition of monocytes, by monocyte culture supernatant, or by the combination of IL1 and IL6. Staining with an anti-IL2 receptor (p55) mAb demonstrated expression of IL2 receptors on about 10% of the anti-CD5-stimulated T cells. Both virgin (CD45RA+) and memory (CD45RO+) T cells were responsive. Our data provide further evidence for the involvement of CD5 in T cell activation.     

CD45 isoforms associated with distinct functions of CD4 cells derived from unusual healthy donors lacking CD45RA- T lymphocytes.

We now report two healthy individuals whose T lymphocytes were over 95% positive for CD45RA antigen expression. However, these donors normally expressed both the CD29 high (CD29+) and CD45RO high (CD45RO+) antigens on approximately 40 and 50% of their CD4 cells, respectively. Despite the strong CD45RA expression on the surface of almost all CD4 cells, the CD29 marker allowed T cells from these donors to be divided phenotypically into subsets having distinct in vitro function. CD4+CD29+ cells from these donors responded maximally to recall antigens such as TT and provided strong helper function for B cell Ig synthesis. In contrast, CD4+CD29- cells responded poorly to recall antigens and had poor helper function for B cell Ig synthesis, but had strong suppressor activity. Thus, CD29 antigen expression was still predictive of the in vitro functional activity as previously described for normal donors. Furthermore, biochemical analysis of the distribution of individual CD45 isoforms on the surface of these subsets of CD4 cells revealed distinct differences. The CD4+CD29 high (CD4+CD29+) subset of cells primarily expressed the 180-, 190-, and 205-kDa CD45 isoforms, while the CD4+CD29 low (CD4+CD29-) cells primarily expressed the 190-, 205-, and 220-kDa CD45 isoforms. These results suggest that despite the superficial phenotypic similarity of CD4 cells in these donors, distinctions in the distribution of both CD29 and the 180- and 220-kDa CD45 isoforms exist and might play a role in the different functions of freshly isolated CD4 lymphocytes.     

Differential interleukin secretion by in vitro activated human CD45RA and CD45RO CD4+ T cell subsets.

The CD45RA and CD45RO isoforms have been reported to define complementary subsets among CD4+ T cells: CD45RA CD4+ T cells are considered "virgin T cells" and CD45RO "primed T cells." We investigated the secretion of lymphokines by human CD4+ CD45RO and CD4+ CD45RA T helper cells after mitogen stimulation. CD45RA and CD45RO CD4+ T cells were isolated by negative immunoselection using magnetic beads. CD45RO cells, but not CD45RA cells, proliferate well in response to pokeweed mitogen (PWM) or insoluble anti-CD3. Both subpopulations produced interleukin (IL)-2, IL-6, and interferon (IFN)-gamma when stimulated with PWM for 1-4 days. Only Day 1 supernatants from CD45RO cells contained moderate amounts of IL-4. After 14 days of continuous culture and stimulation with PWM, the CD45RA subset had lost the expression of CD45RA and gained that of CD45RO. When long-term cultured CD45RA or CD45RO cells were treated with insoluble anti-CD3, they incorporated [3H]thymidine at similar levels, but only CD45RO cells secreted IL-4 and significantly increased their secretion of IFN-gamma. These data indicate that despite phenotype conversion, the two subpopulations maintain functional differences in the secretion of lymphokines, thus suggesting that circulating CD45RA and CD45RO cells may represent different lines of differentiation.     

Human CD8+ T lymphocytes can be divided into CD45RA+ and CD45RO+ cells with different requirements for activation and differentiation.

The functional distinction between CD45RA+ and CD45RO+ cells within the human CD4+ T cell subset is well established. This study was undertaken to investigate whether a similar division can be made within the CD8+ T cell population. A quantitative comparison was made of the requirements for activation and differentiation of CD8+CD45RA+ and CD8+CD45RO+ cells. Stimulation of T lymphocytes with anti-CD3 mAb immobilized at high-density induced strong proliferation and CTL activity in both CD45RA+ and CD45RO+ cells. Suboptimal TCR/CD3 triggering, in contrast, induced substantially higher levels of proliferation and CTL activity in CD8+CD45RO+ cells compared with their CD45RA+ counterparts. Lymphokine secretion (i.e., Il-2 and TNF-alpha) was under any condition more readily induced in CD8+CD45RO+ cells. Markedly, proliferation of both CD8+CD45RA+ and CD8+CD45RO+ T cells initiated by anti-CD3 mAb immobilized at high densities was not inhibited by addition of anti-CD25 mAb, in contrast to proliferation induced by suboptimal anti-CD3 mAb concentrations. These findings show that a functional division between CD45RA+ and CD45RO+ T cells with distinct requirements for activation and differentiation may also be made in the CD8+ subset.     

The coexpression of CD45RA and CD45RO isoforms on T cells during the S/G2/M stages of cell cycle.

The tyrosine phosphatase CD45 is alternatively spliced to generate isoforms of different molecular weights (180-220 kDa) which are differentially expressed on hematopoietic cells. Monoclonal antibodies reacting with either the 180-kDa (UCHL-1, CD45RO) or the 200- to 220-kDa (2H4, CD45RA) isoform have been used to subdivide T cell populations based on their expression of one or the other of these two epitopes. CD45RA T cells have "naive" characteristics of unresponsiveness to recall antigens and prominence in cord blood, while CD45RO T cells are considered "memory" T cells because they proliferate to recall antigens and increase following PHA activation of cord blood. However, we have recently demonstrated the expression of the CD45RA isoform on a subpopulation of CD45RO+ T cell clones, suggesting that CD45RA is not a universal marker for naive T cells. Using propidium iodide staining of the DNA to determine cell cycle stage, we now show that CD45RA expression is significantly higher on T cell clones during the S, G2, and M stages of cell cycle when compared to CD45RA expression on cells in Go and G1. Furthermore, CD45RA expression on cells undergoing mitosis is not limited to long-term activated T cell clones, as uncultured peripheral blood T cells in the S/G2/M phase express significantly more CD45RA. The percentage of T cells coexpressing CD45RA and CD45RO also increases following PHA activation, indicating that T cells in the process of division express both isoforms. These results suggest a potential role of the CD45RA isoform during the stages of cell cycle leading to mitosis.     

Identification of the anti-CD3-unresponsive subpopulation of CD4+, CD45RA+ peripheral T lymphocytes.

The majority of peripheral CD4+ T lymphocytes proliferate in vitro in response to anti-CD3 in presence of autologous APC. The present study describes a subpopulation of CD4+ T cells that cannot be activated and progress into cell cycle by stimulation with anti-CD3 plus APC or with mitogenic combinations of anti-CD2. The in vitro responses of these anti-CD3-unresponsive CD4+ T cells were investigated with a panel of mAb to CD2, CD3, and CD28, and found to be similar to those previously observed for mature thymocytes: only the combination of anti-CD2 plus anti-CD28 produced cell proliferation. Anti-CD3-unresponsive T cells were CD45RA+, but represented only 14 to 22% of the CD4+, CD45RA+ T cell population. Activation with anti-CD2 plus anti-CD28 mAb resulted in major changes in the cell surface phenotype and functional properties: a loss of CD45RA+ occurred and an increased expression of CD45RO, CD29, and CD58 (LFA3), as well as a gain in responsiveness to anti-CD3 and anti-CD2. This change in CD45 phenotype from CD45RA to CD45RO occurs in both the anti-CD3-responsive and in the anti-CD3-unresponsive subsets of the CD45RA+, CD4+ cells after cell proliferation. The anti-CD3-unresponsive subset may represent a pool of not yet fully differentiated peripheral T cells. The acquisition of anti-CD3 responsiveness could occur as a consequence of Ag priming or by an Ag-independent mechanism. Involvement of the CD28 Ag in this process is suggested from the present study.     

CD27, a member of the nerve growth factor receptor family, is preferentially expressed on CD45RA+ CD4 T cell clones and involved in distinct immunoregulatory functions.

CD27 is a disulfide-linked 120-kDa homodimer expressed on the majority of peripheral T cells at variable density that belongs to the recently defined nerve growth factor receptor family. mAb reactive with CD27 can either enhance or inhibit T cell activation, suggesting a crucial role in the process of T cell activation. We now show that CD27 is preferentially expressed on the CD45RA+CD45RO-CD29low subset of CD4 cells. CD27 expression on this subset is maintained for a prolonged period in culture after PHA activation. In contrast, CD45RA-CD45RO(+)-CD29high subset of CD4 cells express very low level of CD27, and its expression is lost within 2 wk after PHA activation. To further analyze the differential expression of CD27 on these reciprocal subsets of CD4 cells, we developed T cell clones by stimulating isolated CD4+CD45RA+ and CD4+CD45RO+ populations with PHA. T cell clones derived from cells originally CD45RA+ retained both CD45RA and CD27 expression, whereas T cell clones derived from cells originally CD45RO+ were CD45RA- and CD27-. In functional assays, IL-4 production could only be induced in CD45RA-CD27- CD4 clones by stimulation with PMA and ionomycin. Four of six CD45RA+ CD4 clones had suppressor activity in PWM-driven IgG synthesis, whereas five of six CD45RA- CD4 clones had helper activity. Of interest, the suppressor activity of CD45RA+CD27+ clones was partially blocked by pretreatment with anti-CD27 mAb (1A4). Anti-1A4 pretreatment of these T cell clones resulted in elevation of intracellular cAMP levels. Thus, CD27 appears to play a role in the function of CD45RA+CD27+ CD4 cells, and may be involved in suppressor activity of these cells at least in part via its effects on cAMP production.     

Production of tumor necrosis factor-alpha by naive or memory T lymphocytes activated via CD28.

While it is well established that activated T cells can produce tumor necrosis factor alpha (TNF-alpha), it is less clear whether this function is confined to a given subset, e.g., memory cells. To approach this question, we investigated the production of TNF-alpha by human peripheral blood T lymphocytes activated with anti-CD28 mAb since this activation pathway is known to potentiate cytokine production. Under the culture conditions used, the amount of TNF-alpha produced was markedly enhanced compared to that obtained after activation with immobilized anti-CD3 mAb. The enhancement of TNF-alpha production was already apparent after incubation of T cells for 6 hr. Up to 5 ng/ml of TNF-alpha was measured on Day 2 in supernatants of cultures of 10(4) T lymphocytes. To determine the source of the cells producing high amounts of TNF-alpha, T lymphocytes were separated into two subpopulations, namely naive cells (expressing the CD45RA isoform) and memory cells (expressing the CD45RO isoform). While both subpopulations proliferated equally well after stimulation with anti-CD28 mAb, up to 90% of the TNF-alpha produced under these conditions originated from memory T cells. These results thus document that T cell activation via CD28 results in a marked increase in TNF-alpha production without affecting the functional disparity that exists between naive and memory T cells.     

Development of a monoclonal antibody, anti-6C2, which is involved in the interaction of CD4 T helper cells and activated B cells.

We have developed a mAb anti-6C2, by immunizing mice with T cell line derived from the Callithrix jacchus (common marmoset). Anti-6C2 is reactive with approximately 50% of unfractionated T cells, 50% of CD4+ cells, and 40% of CD8+ cells. Regarding CD4+ cells, anti-6C2-reactive cells substantially overlap with the CD29+CD45RO+ Th cell population. Moreover, anti-6C2 can divide these T cells into 6C2+ and 6C2- subpopulations. The CD4+CD45RO+6C2+ cells maximally respond to soluble Ag such as tetanus toxoid and provide strong helper function for PWM-driven B cell IgG synthesis. Most interestingly, anti-6C2 was also reactive against activated B cells but not resting B cells; furthermore, this epitope was inducible through activation of resting B cells or B cell line. Biochemical characterization showed that anti-6C2 precipitated two glycoproteins with the relative molecular weights of 180,000 and 95,000 from 125I-surface labeled cell lysate. Sequential immunoprecipitation studies demonstrated that these two glycoproteins were the lymphocyte function-associated antigen (LFA-1) Ag complex (CD11a/18). Significantly, although this antibody did not inhibit cytotoxic killer T cell responses and Ag-induced T cell proliferation as did conventional anti-LFA-1, it did inhibit PWM-driven B cell IgG synthesis. Because 6C2 expression was induced after B cell activation, the above results strongly suggest that the 6C2 molecule may play a role in the interaction of CD4 helper cells and activated B lymphocytes.     

Biochemical nature and topographic localization of epitopes defining four distinct CD45 antigen specificities. Conventional CD45, CD45R, 180 kDa (UCHL1) and 220/205/190 kDa

The biochemical nature and relative topographic localization of Ag determinants recognized on CD45 molecular complex by mAb defining four distinct Ag specificities (conventional CD45, CD45R, 180 kDa and 220/205/190 kDa) have been investigated. These Ag specificities display a differential biochemical, cellular, and histochemical distributions and are important in the definition of CD4-positive complementary functional T cell subsets and/or distinct stages of thymic maturation. Protease treatment of either CD45-positive cells or purified CD45 molecules revealed that both conventional CD45 and 180-kDa (UCHL1 epitope) Ag specificities are defined by epitopes present on a protease-resistant domain which is internal to the protease-sensitive epitopes defining both CD45R and 220/205/190-kDa Ag specificities. In addition, it is shown that carbohydrate moieties are contributing to the epitopes recognized by both the anti-180-kDa UCHL1 and the anti-220/205/190-kDa mAb. Neuraminidase treatment, which cleaves sialic acids either from N- or O-linked oligosaccharides, abrogated the reactivity of both mAb. However, N-glycanase treatment, which selectively cleaves N-linked sugars, did not affect the recognition of these two epitopes. Thus, these results demonstrate that the Ag determinants recognized by the UCHL1 and the anti-220/205/190-kDa mAb, which are topographically unrelated, are associated with sialic acids from O-linked-type oligosaccharides, emphasizing the contribution of carbohydrates to the Ag heterogeneity of CD45 molecular complex.     

Nuclear factor of activated T cells is a driving force for preferential productive HIV-1 infection of CD45RO-expressing CD4+ T cells

Human immunodeficiency virus type-1 (HIV-1) preferentially replicates in CD4-expressing T cells bearing a "memory" (CD45RO+) rather than a "naive" (CD45RA+/CD62L+) phenotype. Yet the basis for the higher susceptibility of these cells to HIV-1 infection remains unclear. Because the nature of the CD45 isoform itself can affect biochemical events in T cells, we set out to determine whether these isoforms could differently modulate HIV-1 long terminal repeat (LTR) activity and thereby replication. Through the use of CD4+ Jurkat T cells specifically expressing distinct CD45 isoforms (i.e. CD45RABC or CD45RO), we demonstrated that a difference in CD45 isoform expression conferred preferential replication of HIV-1 to CD45RO-expressing T cell clones following a physiological CD3/CD28 stimulation. Closer analysis indicated that higher HIV-1 LTR activation levels were consistently observed in CD45RO-positive cells, which was paralleled by more pronounced nuclear factor of activated T cells (NFAT) activation in these same cells. Specific involvement of NFAT1 was revealed in studied Jurkat clones by mobility shift analyses. In addition, preferential activation of the LTR and viral replication in CD45RO T cells was FK506- and cyclosporin A-sensitive. These results underscore the importance of NFAT in HIV-1 regulation and for the first time identify the role of the CD45 isoform in limiting productive HIV-1 replication to the human CD4 memory T cell subset.     

1F7, a novel cell surface molecule, involved in helper function of CD4 cells

We have developed a monoclonal antibody, anti-1F7, that inhibits soluble Ag-driven T cell proliferation as well as PWM-driven IgG synthesis. Anti-1F7 antibody reacts with approximately 57% of unfractionated T cells, 62% of CD4+ cells, and 54% of CD8+ cells. Although the 1F7 Ag is widely distributed among lymphoid cells, this Ag on CD4+ cells is preferentially expressed on the CDw29(4B4+) helper population. Moreover, anti-1F7 antibody further subdivides the CD4+CDw29+ cell subset into CDw29+1F7+ and CDw29+1F7- populations. The CD4+CDw29+1F7+ population of cells maximally proliferates to recall Ag such as tetanus toxoid, whereas helper function for PWM-driven IgG synthesis by B cells belongs to both the CD4+CDw29+1F7+ and CD4+CDw29+1F7- population of cells. The most prominent structure defined by this antibody is a 110-kDa molecule that is different from the 135-kDa, 160-kDa, and 185-kDa glycoproteins identified by anti-CDw29 antibody and the 180-kDa glycoprotein identified by UCHL-1 antibody. It is, however, related to the molecule recognized by anti-Ta1, an activation Ag on T cells. Furthermore, although the Ta1 molecule is recognized by anti-1F7 mAb, the 1F7 family of structures also includes molecules distinct from Ta1.     

Prolonged expression of high molecular mass CD45RA isoform during the differentiation of human progenitor thymocytes to CD3+ cells in vitro.

CD45, the leukocyte common Ag, has been shown to characterize T cell development both within the thymus and among peripheral T cells. The work reported here demonstrates that human multinegative (MN) thymocytes, depleted of cells bearing CD3, CD4, CD8, and CD19, express predominantly the high molecular mass CD45RA isoform, and lack low molecular mass CD45RB isoforms and CD45R0 as detected by immunofluorescence. By immunoprecipitation of surface-labeled CD45 molecules from MN thymocytes, a proportion of the CD45 is in fact of low molecular mass but does not include epitopes recognized by CD45R0, nor by CD45RB mAb specific for the p190. This suggests either glycosylation variants of CD45RB/CD45R0 undetectable by our mAb, or underglycosylated CD45RA. MN thymocytes lack TCR-alpha beta mRNA confirming their early developmental stage. Upon culture with IL-2 or with mitogenic combinations of anti-CD2/CD28 mAb, MN thymocytes differentiate to acquire CD3, TCR-alpha beta, and in some cases CD4 and/or CD8. We have predicted that maintenance of CD45RA and lack of CD45R0 expression is fundamental to generative thymic development. If correct, this demands that unlike peripheral T cells, differentiation of MN thymocytes should be accompanied by prolonged expression of high molecular mass CD45 isoforms. Analysis of CD45 isoform expression during MN thymocyte development confirms this prediction and indicates that expression of CD45RA is maintained, at increasing density, for at least 8 to 12 days of culture. Unlike peripheral blood T cells, this is accompanied by the gradual acquisition of firstly the p190 isoforms of CD45RB and later by CD45R0, resulting in a population of CD3+TCR-alpha beta cells coexpressing CD45RA/RBp190/R0. Dot blot analysis of mRNA from differentiating MN thymocytes indicates prolonged expression of mRNA encoding CD45 exons a, b, and c, again in contrast to peripheral T cells which lose all mRNA for alternatively spliced CD45 exons within the first 24 h poststimulation. This is discussed in the context of negative selection during thymic development and interconversion of T cell subsets.     

Expression characteristics and stimulatory functions of CD43 in human CD4+ memory T cells: analysis using a monoclonal antibody to CD43 that has a novel lineage specificity

We have used HSCA-2, an mAb that recognizes a sialic acid-dependent epitope on the low molecular mass (approximately 115-kDa) glycoform of CD43 that is expressed in resting T and NK cells, to examine the expression characteristics and stimulatory functions of CD43 in human CD4+ memory T cells. Having previously reported that the memory cells that respond to recall Ags in a CD4+ CD45RO+ T cell population almost all belong to a subset whose surface CD43 expression levels are elevated, we now find that exposing these same memory T cells to HSCA-2 mAb markedly increases their proliferative responsiveness to recall Ags. We think it unlikely that this increase in responsiveness is a result of CD43-mediated monocyte activation, especially given that the HSCA-2 mAb differs from all previously used CD43 mAbs in having no obvious binding specificity for monocyte CD43. Predictably, treatment with HSCA-2 mAb did not lead to significant recall responses in CD4+ CD45RO+ T cells, whose CD43 expression levels were similar to or lower than those of naive cells. Other experiments indicated that the HSCA-2 mAb was capable of enhancing the proliferative responsiveness of CD4+ memory T cells that had been exposed to polyclonal stimulation by monocyte-bound CD3 mAb and could also act in synergy with CD28 mAb to enhance the responsiveness of CD4+ T cells to CD3 stimulation. Taken together, these findings suggest that the CD43 molecules expressed on CD4+ memory T cells may be capable of enhancing the costimulatory signaling and hence providing accessory functions to TCR-mediated activation processes.     

Immobilized anti-CD3-induced T cell growth: comparison of the frequency of responding cells within various T cell subsets.

Human T cells can be divided into subsets based on the expression of CD29, CD45RA, CD45RO, LFA-3, or CD11a. It has been suggested that the subset of CD4+ T cells that expresses high densities of CD29, CD11a, CD45RO, and LFA-3 contains "memory" T cells, whereas the subset of cells that expresses CD45RA contains "naive" T cells. In order to obtain a more complete picture of the functional capacities of human naive and memory CD4+ and CD8+ T cell subsets, highly purified T cells were activated with a uniform stimulus and responses were examined in bulk cultures and under limiting dilution conditions. T cell activation was achieved with an immobilized mAb to the CD3 molecular complex, 64.1. In bulk cultures, immobilized 64.1 stimulated a vigorous response. Moreover, the number of cells entering the cell cycle, the magnitude of the [3H]thymidine incorporation, and the growth of the cells over 6 days in culture by naive and memory CD4+ T cells was comparable. To delineate the frequency of responsive cells in each subset more precisely, cells were cultured with immobilized 64.1 at limiting dilution and the precursor frequency of responding cells was assessed by examining wells microscopically for visible growth. Immobilized 64.1 was able to induce some T cells from each subset to grow in the complete absence of AC, when exogenous IL2 was present. The number of responding CD4+ and CD8+ cells was comparable. The percentage of naive cells responding in each population was approximately three times greater than the frequency of memory cells. IL4 could also support the growth of immobilized 64.1-activated CD4+ T cells, but the frequency of responding cells was much lower than that supported by IL2. The vast majority of the IL-4 responsive CD4+ cells resided within the naive cell subset. The data indicate that the response of CD4+ and CD8+ naive and memory T cell subsets to immobilized anti-CD3 depends on the density of responding cells. Naive T cells have an enhanced capacity to grow when cultured in the absence of other T cells or accessory cells. This ability may facilitate their expansion during primary immune responses.     

The functional heterogeneity of CD8+ cells defined by anti-CD45RA (2H4) and anti-CD29 (4B4) antibodies.

The monoclonal antibodies, anti-2H4(CD45RA), and anti-4B4(CD29), along with UCHL1-(CD45RO), identify reciprocal populations of CD4 cells with distinct suppressor inducer (CD45RA+CD29-CD45RO-) and helper inducer (CD45RA-CD29+CD45RO+) functions. Although the CD8+ population is known to contain precytotoxic, cytotoxic, suppressor, and some natural killer cells, the exact phenotypic identities of these functional CD8 subsets has not been established. In this study, we tried to determine whether these monoclonal antibodies could distinguish functionally distinct subsets of cells within the CD8+ population. For this purpose, whole T cells or fractionated T cells were sensitized with irradiated allogeneic non-T cells for 6 days, following which, CD8+ or CD8+CD11b- cells were isolated and cellular functions such as suppressor, killer precursor, and killer effector activity were assessed. The results showed that both class I-restricted alloantigen-specific killer effector and killer precursor cells belonged to the CD8+CD11b-CD45RA-CD29+ population. Moreover, these killer effector cells expressed the CTL-associated S6F1 molecule, an epitope of the LFA-1 antigen. In contrast, suppressor effector cells belonged to the CD8+CD11b-CD45RA+CD29- cell population. Although the UCHL1 antigen has been reported to define the CD4+CD29+ helper inducer cell, over 90% of allo-activated CD8+ cells expressed this antigen, whereas only 40-60% of these cells expressed either CD45RA or CD29 antigens. These results suggest that anti-CD45RA and anti-CD29 antibodies may provide useful tools for distinguishing between suppressor effector versus killer effector and killer precursor cells within the CD8+CD11b- population.     

T cell regulation of human B cell proliferation and differentiation. Regulatory influences of CD45R+ and CD45R- T4 cell subsets

The immunoregulatory functions of human T4 cell subpopulations defined by mAb to the CD45R molecule (2H4) were examined. Both CD45R- and CD45R+ T4 cells that had been treated with mitomycin C (CD45R- and CD45R+ T4-mito) provided help for the generation of Ig-secreting cells (ISC) in cultures stimulated by PWM or by immobilized mAb to CD3 (64.1). IL-2 enhanced the generation of ISC in PWM-stimulated cultures and in anti-CD3-stimulated cultures containing CD45R+ T4-mito. The generation of ISC was maximal in cultures containing anti-CD3-activated CD45R- T4-mito and was not increased by IL-2. By contrast, CD45R+ T4 cells that had not been treated with mitomycin C suppressed B cell responses in cultures stimulated with PWM or anti-CD3, whereas CD45R- T4 cells suppressed the generation of ISC only in cultures stimulated with anti-CD3. IL-2 enhanced suppression by anti-CD3, but not PWM, activated CD45R- T4 cells. Suppression by CD45R+ T4 cells was maximal and not increased by IL-2. CD45R+ T4-mito were more effective suppressor-inducers in PWM-stimulated cultures, promoting the differentiation of suppressor-effector cells from CD8+ T cells. However, both CD45R+ and CD45R- T4-mito exerted comparable suppressor-inducer function in anti-CD3-stimulated cultures. Moreover, in anti-CD3-stimulated cultures, T8 cells could function as both suppressor-effector cells and suppressor-inducer cells. One of the functions of suppressor-inducer cells in this system appeared to involve the production of IL-2. Thus, the addition of IL-2 facilitated the induction of suppressor-effector T8 cells by CD45R- T4-mito in PWM-stimulated cultures. Although IL-2 production by the T cell subsets varied widely depending on the nature of the stimulus, these differences could not entirely explain their capacity to function as helper cells, suppressor-effector cells or suppressor-inducer cells. These results indicate that both CD45R+ and CD45R- T4 cells can help or suppress B cell responses, as well as induce suppressor-effector T8 cells. Moreover, suppressor-inducer function of T cells is not limited to the T4 cell population, but rather can also be accomplished by T8 cells. The results indicate that both T4 cell subsets and T8 cells exert multiple regulatory effects on human B cell function, with the nature of the activating stimulus playing a major role in determining the functional capacity of various T cell subsets.     

Stimulation via the CD3 and CD28 molecules induces responsiveness to IL-4 in CD4+CD29+CD45R- memory T lymphocytes

Although both IL-2 and IL-4 can promote the growth of activated T cells, IL-4 appears to selectively promote the growth of those helper/inducer and cytolytic T cells which have been activated via their CD3/TCR complex. The present study examines the participation of CD28 and certain other T cell-surface molecules in inducing T cell responsiveness to IL-4. Purified small high density T cells were cultured in the absence of accessory cells with various soluble anti-human T cell mAb with or without soluble anti-CD3 mAb and their responsiveness to IL-4 was studied. None of the soluble anti-T cell mAb alone was able to induce T cell proliferation in response to IL-4. A combination of soluble anti-CD3 with anti-CD28 mAb but not with mAb directed at the CD2, CD5, CD7, CD11a/CD18, or class I MHC molecules induced T cell proliferation in response to IL-4. Anti-CD2 and anti-CD5 mAb enhanced and anti-CD18 mAb inhibited this anti-CD3 + anti-CD28 mAb-induced T cell response to IL-4. In addition, anti-CD2 in combination with anti-CD3 and anti-CD28 mAb induced modest levels of T cell proliferation even in the absence of exogenous cytokines. IL-1, IL-6, and TNF were each unable to replace either anti-CD3 or anti-CD28 mAb in the induction of T cell responsiveness to IL-4, but both IL-1 and TNF enhanced this response. The anti-CD3 + anti-CD28 mAb-induced response to IL-4 was exhibited only by cells within the CD4+CD29+CD45R- memory T subpopulation, and not by CD8+ or CD4+CD45R+ naive T cells. When individually cross-linked with goat anti-mouse IgG antibody immobilized on plastic surface, only anti-CD3 and anti-CD28 mAb were able to induce T cell proliferation. These results indicate that the CD3 and CD28 molecules play a crucial role in inducing T cell responsiveness to IL-4 and that the CD2, CD5, and CD11a/CD18 molecules influence this process.     

CD31, a novel cell surface marker for CD4 cells of suppressor lineage, unaltered by state of activation.

In this study, we examined the role of CD31 as a cell surface marker for subsets of human CD4 cells. CD31, as defined by a newly developed mAb termed anti-1F11, can divide activated as well as resting CD4 cells into distinct functional subpopulations, based on its surface expression. Among CD4 cells freshly isolated from peripheral blood, anti-1F11 preferentially reacts with the CD45RA+ subset. The majority of helper activity for B cell IgG synthesis and memory function to recall Ag such as tetanus toxoid or mumps was found within the CD31- CD4 cell population, whereas CD31+ CD4 cells provided poor helper function for B cell IgG synthesis and were more responsive to Con A and autologous MHC (autologous MLR). The expression of CD31 on CD45RA+ CD4 cells did not change after activation, despite the loss of CD45RA from the cell surface. Conversely, CD31 was not acquired after activation of CD45RO+ CD45RA- CD4 cells. Furthermore, activated CD4 cells expressing CD31 can induce suppressor function for B cell IgG synthesis, whereas the reciprocal population of activated CD4 cells (CD31-) provide strong helper function for B cell IgG production. Finally, IL-4 production could only be induced by stimulation with PMA and ionomycin in either resting or activated CD31- CD4 cells. Thus, CD31 may prove useful in defining CD4 populations with reciprocal functional programs. Moreover, unlike other markers used for this purpose, the expression of CD31 does not change after activation and may serve as a more useful marker for identification of cells of suppressor or helper lineage.     

Human peripheral blood T helper cell-induced B cell activation results in B cell surface expression of the CD23 (BLAST-2) antigen

We have developed an in vitro system to assess the early stages of B cell activation induced by peripheral blood T helper cells. Peripheral blood mononuclear cells are cultured for 16 hr with anti-CD3 monoclonal antibody (mAb), T lymphocytes are then removed by sheep red blood cell rosette depletion, and expression of the B cell surface activation antigen CD23 (BLAST-2) is assessed by indirect immunofluorescence. Anti-CD3 mAb, but not a control anti-CD5 mAb, stimulates the expression of CD23 on 20-50% of peripheral blood B cells cultured with autologous T cells. T cell subset depletion studies show that the CD4+ T cell subset is responsible for anti-CD3-mediated induction of CD23 on autologous B cells. Anti-CD3-induced, T helper cell-dependent CD23 expression is not MHC-restricted, as allogeneic combinations of T and non-T cells, cultured in the presence of anti-CD3 antibody, also result in the expression of B cell CD23. Individuals whose monocyte Fc receptors bind murine IgG1 mAb poorly fail to trigger T cell proliferation in response to murine IgG1 anti-CD3 mAb and also fail to express B cell CD23 following culture of PBMC with IgG1 anti-CD3 mAb, while the usual expression of CD23 is seen after culture with IgG2a anti-CD3 mAb. The mechanism of anti-CD3-induced B cell activation was addressed in experiments using a two-chamber culture system. While little IL-4 activity was detected in anti-CD3-stimulated culture supernatants, optimal induction of CD23 was observed when T and B cells were cultured together in a single chamber. This suggests that under physiologic conditions, in which quantities of lymphokine may be limiting, close physical contact between the anti-CD3-activated Th cell and B cell may be required for CD23 expression. The anti-CD3-induced BLAST-2 assay will facilitate the analysis of Th cell-mediated B cell activation in any individual and should permit us to separately evaluate the roles of Th cells and B cells in the impaired immunoregulation characteristic of autoimmune disorders.