Different epitopes of the CD31 antigen identified by monoclonal antibodies: cell type-specific patterns of expression

3 mAb-5A2.G5, B2B1 and 2BD4-all of IgG1 isotype were identified as belonging to the CD31 cluster by their binding to transfected murine cell lines expressing the CD31 antigen and by sequential immunoprecipitation experiments. Competitive binding experiments were carried out using the human myelomonocytic cell line RC-2A. mAb B2B1 and 2BD4 did not cross block. mAb 5A2.G5 partly inhibited binding of 2BD4 but not B2B1. Thus the epitopes identified by 5A2.G5 and 2BD4 appear to overlap but to be quite separate from that recognized by B2B1. All 3 antibodies bound to a 130 kDa species on Western blots after nonreducing polyacrylamide gel electrophoresis of platelet lysates. Culture of RC-2A cells in the presence of tunicamycin (after removal of surface antigens by pronase) blocked re-expression of the epitopes recognised by all 3 mAb suggesting that they involve N-linked glycosylation. Furthermore, treatment of platelet lysates with Endoglycosidase F prior to electrophoresis and Western blotting abolished the binding of the mAb but not a rabbit polyclonal antiserum to CD31. Nevertheless, neuraminidase treatment of RC-2A cells failed to affect mAb binding. The antibodies displayed typical properties of CD31 antibodies in that all 3 precipitated at 130 kDa cell surface protein and bound strongly to platelets, monocytes, neutrophils and vascular endothelium. All 3 antibodies were positive on hemopoietic progenitor cells which give rise to colonies in the CFU-C assay. However, differences in binding to peripheral blood lymphocytes and to certain human leukemic cell lines were noted. In particular, mAb 5A2.G5 bound weakly to lymphocytes and to the lymphoid cell line HSB-2 compared with the other 2 antibodies.     

Flow Cytometric Identification of CD93 Expression on Naive T Lymphocytes (CD4<Superscript>+</Superscript>CD45RA<Superscript>+</Superscript> Cells) in Human Neonatal Umbilical Cord Blood

Human CD93 has a molecular weight of about 100 kDa and is selectively expressed by myeloid cell lineages in peripheral blood (PB) mononuclear cells. Although CD93 was initially identified as a receptor for complement component 1, subcomponent q phagocytosis (C1qRp) involved in the C1q-mediated enhancement of the phagocytosis of various antigens, several recent studies have reported that CD93 is not a receptor for the C1q-mediated enhancement of phagocytosis. The expression patterns of CD93 have been previously investigated in PB mononuclear cells (lymphocytes, monocytes, and granulocytes) from adult PB and neonatal umbilical cord blood (UCB), and the expression of CD93 was not found on lymphocytes from either normal adult PB or neonatal UCB. However, the detection of CD93 expression in neonatal UCB using CD93 monoclonal antibodies (mAbs) that recognize different antigenic epitopes remains poorly understood. In this study, we examined the expression of CD93 on lymphocytes, monocytes, and granulocytes from neonatal UCB using four different types of CD93 mAb detection probes, mNI-11, R139, R3, and X-2, using flow cytometric and western blot analyses. We found that CD93, as defined using all four mAbs, was expressed on monocytes and granulocytes in PB mononuclear cells from adult PB and neonatal UCB. On the other hand, we observed for the first time that the expression of CD93 on lymphocytes in neonatal UCB can only be detected using the mNI-11 mAb, established in our laboratory, and not with commercially available CD93 mAbs (R139, R3, and X-2). However, CD93 expression on lymphocytes from normal adults was not detected using any of the four CD93 mAbs. Two-color flow cytometric analyses showed that the CD93 recognized by mNI-11 mAb was expressed on CD3⁺ T lymphocytes (mainly CD4⁺ helper T lymphocytes), but not on CD19⁺ B lymphocytes or on CD8⁺ suppressor/cytotoxic T lymphocytes from neonatal UCB. In addition, CD93 was expressed on CD45RA⁺ (naive antigen) lymphocytes from neonatal UCB, but not on CD45RO⁺ (memory antigen) lymphocytes from neonatal UCB or on CD45RA⁺ and CD45RO⁺ lymphocytes from normal adult PB. Three-color flow cytometric analysis showed that CD93 was co-expressed on naive T lymphocytes (CD4⁺CD45RA⁺) from neonatal UCB. In a western blot analysis, the CD93 mAb (mNI-11) immunoprecipitated at a molecular weight of 98 kDa, identified as a CD93 molecule, in the CD4⁺CD45RA⁺ cells from neonatal UCB but not from adult PB, similar to the results in the human monocyte-like cell line U937 (human CD93-positive cells). Taken together, these results provide the first direct evidence of a novel/naive cell population (CD4⁺CD45RA⁺CD93⁺) in neonatal UCB that may have an important role in cell biology, transplantation, and immature/mature immune responses.     

Identical expression of CD45R isoforms by CD45RC+ 'revertant' memory and CD45RC+ naive CD4 T cells.

Naive and memory CD4 T cells are frequently defined by exon-specific monoclonal antibodies (mAb) which stain (or not) high- or low-molecular-weight (MW) isoforms of the leucocyte common antigen CD45. The link between isoform and the naive/memory designation is complicated by the fact that CD4 T cells with a 'memory' phenotype (CD45RA-, RB-, RC-, or CD45RO+) may revert ('revertants') and re-express the high mw isoform (CD45RA+, RB+, RC+). Isoform expression also changes during normal T-cell development. Furthermore, the picture may be incomplete since an exon-specific mAb will not detect all possible isoforms on a cell. We have used molecular techniques to determine whether revertant CD4 memory T cells were different from naive T cells with respect to CD45R isoform expression. Using the anti-CD45RC mAb OX22 to purify rat lymphocyte subsets, CD45R isoform expression was examined at the mRNA level in CD4 T cells at different stages of development and compared with that of B cells and unseparated lymphocytes. B cells contained abundant message for the highest MW 3-exon isoform ABC, the 2-exon isoforms AB and BC, and the null isoform O. Both immature CD45RC- (i.e. CD4+8- 'single positive' thymocytes, and peripheral Thy-1+ recent thymic emigrants) and mature CD45RC- 'antigen-experienced' CD4 T cells had message for single-exons B, possibly C and for the O exon. In contrast, CD45RC+ CD4 T cells contained mRNA coding for ABC (low level), AB, BC, B, C (low level) and O (low level). Importantly, there was no difference between CD45RC+ T cells that had not seen antigen ('truly native') and CD45RC+ antigen-experienced revertant memory T cells. This observation has implications for understanding long-term immunological memory.     

Ligation of CD31/PECAM-1 modulates the function of lymphocytes,monocytes and neutrophils

CD31 or platelet/endothelial cell adhesion molecule (PECAM-1) is a 130-kDa glycoprotein expressed on endothelial cells, granulocytes, a subset of lymphocytes and platelets. In this study, we examined the ability of four monoclonal antibodies (mAb) against different domains of CD31 to modulate the function of T lymphocytes, monocytes and neutrophils. Engagement of CD31 on T lymphocytes results in co-stimulation of T lymphocyte proliferation to suboptimal doses of anti-CD31 mAb. This proliferation is accompanied by secretion of numerous cytokines and chemokines, up-regulation of CD25 and an increase in cell size. Purification of T lymphocytes into CD45RO and CD45RA subsets showed that only naive CD45RA T lymphocytes are co-stimulated by anti-CD31 mAb. Further studies on neutrophils show that engagement of CD31 results in down-regulation of CD62L and up-regulation of CD11b/CD18 as well as oxidative burst, as assessed by superoxide release. In addition, ligation of CD31 on monocytes results in TNF-α secretion, and studies with various cell signaling inhibitors indicate that tyrosine kinases and cAMP-dependent kinases are involved in monocyte activation via CD31. Of the four mAb used in this study, only two activated human leukocytes. These mAb were PECAM-1.3 and hec7, which bind to domains 1 and 2 of CD31. We conclude that engagement of domains 1 and 2 of CD31 results in outside-in signaling in leukocytes.     

CD3+ WT31- peripheral T lymphocytes lack T44 (CD28), a surface molecule involved in activation of T cells bearing the alpha/beta heterodimer

We have applied two-color fluorescence cytofluorometric techniques to the analysis of the distribution of T44 and CD3 antigens in peripheral blood human lymphocytes. While most CD3+ cells co-expressed T44 antigen, a small distinct subset was CD3+ T44- (2-10% of CD3+ cells). This cell subset also did not react with the WT31 monoclonal antibody (mAb), specific for an alpha/beta framework determinant of the T cell receptor (TCR). Lack of T44 antigen expression was also observed in purified CD3+ WT31- polyclonal populations that had been cultured in medium containing interleukin 2 (IL2) and as well as greater than 30 clones expressing the CD3+4-8-WT31- surface phenotype. Immunoprecipitation experiments confirmed that expression of T44 molecules was confined to CD3+ WT31+ peripheral blood T cells. While conventional CD3+ WT31+ cells produced IL2 in response to mAb directed to CD2, CD3 or T44 surface molecules, CD3+ WT31- cells did not respond to anti-T44 mAb but released IL2 following stimulation with anti-CD2 or anti-CD3 mAb. Therefore, assuming that anti-T44 mimicks the effect of a still undefined natural ligand our data suggest that T cells expressing the gamma-gene surface product may be signalled by stimuli which differ, at least in part, from those acting on CD3+ WT31+ T lymphocytes.     

A lymphocyte differentiation and activation antigen, CZ-1, that distinguishes between CD8+ and unstimulated CD4+ T lymphocytes.

We report the generation and cellular reactivity of a novel rat IgM monoclonal antibody (mAb), CZ-1, made against mouse natural killer (NK) cells activated in vivo. mAb CZ-1 recognizes a molecule whose properties are consistent with that of a trypsin-sensitive, non-phosphatidyl inositol-linked sialoglycoprotein. The expression of the antigen recognized by mAb CZ-1 is restricted mostly to cells of the lymphoid lineage. The antigen is expressed on 10%-25% of bone marrow cells and 3%-5% of thymocytes. Analysis of thymocyte subpopulations indicates expression of the CZ-1 antigen on 100% of the NK1.1+, 27% of the CD4-CD8-, 1.1% of the CD4+CD8+, 1.1% of the CD4+CD8-, and 33% of the CD4-CD8+ cells. In the spleen, the CZ-1 antigen is expressed on B lymphocytes, NK cells, and virtually all CD8+ T lymphocytes. Most unstimulated CD4+ splenic T lymphocytes, monocytes and polymorphonuclear cells, with the notable exception of basophils, do not react with mAb CZ-1. CD4+ T cells activated in vivo by virus infection or in vitro by anti-CD3 and interleukin-2 express the CZ-1 antigen. These results indicate that mAb CZ-1 identifies a novel inducible lymphocyte activation/differentiation antigen that distinguishes between thymic and unstimulated splenic CD4+ and CD8+ T lymphocytes. This mAb will be a useful tool in the identification of lymphocyte subpopulations and in the study of the ontogeny and activation of these cells.     

Engagement of CD45 during in vitro priming enhances antigen-specific Th cell frequencies

CD45 is a transmembrane protein tyrosine phosphatase expressedby all lymphoid cells including T cells. Substantial experimentaldata has shown that CD45 maintains a permissive state for TCRsignaling. The highly glycosylated extracellular domain of CD45may be the site of Interaction with regulatory lectin-like counter-receptorson antigen-presenting cells. The mAb NDA5, recognizing a uniquebut broadly distributed epitope of CD45, was used to study thepossible immunoregulatory role of CD45 during antl-CD3 and antigen-specificCD4⁺ T cell activation. In vitro priming of peripheral bloodmononuclear cells with peptlde antigens in the presence of mAbNDA5 results in a higher frequency of antigen-specific T cells.The responses of both naive and memory T cells to peptide antigenswere sensitive to mAb NDA5-enhanced priming. Anti-CD3 activationof normal resting T cells, in the presence of mAb NDA5, resultedIn enhancement of tyrosine phosphorylation of specific intracellularproteins associated with TCR signal transduction. In cultureswithout antigen, mAb NDA5 down-regulated the cell surface expressionof both CD3 and CD4, yet did not stimulate proliferation ofresting T cells. Together these results suggest that engagementof CD45 during in vitro priming has a significant effect onthe development of antigen-specific T cell populations.     

Both CD45R(low) and CD45R(high) "revertant" CD4 memory T cells provide help for memory B cells

During a primary response to thymus dependent antigens, B cells undergo a number of qualitative changes to become memory B cells - processes that require co-stimulatory signals and cytokine help from CD4 T cells. The question of whether distinct, antigen-experienced memory CD4 T cells are subsequently needed to program memory B cells into antibody synthesis has not been clearly resolved.Using an adoptive transfer model in which memory but not naive B cells were stimulated, we evaluated CD4 T cell help using lymphocytes obtained from primed or unprimed thymectomized donors and expressing a naive (CD45R(high)) or a memory (CD45R(low)) phenotype. Memory B cells, most of which were committed to the IgG1 (Th2) subclass, could be stimulated to produce antibody using help transferred by the CD45R(high) naive subset of unprimed donors (slow onset of response), the CD45R(low) subset of 7 day primed donors (large, rapid antibody response) or by both the CD45R(low) and the CD45R(high) "revertant" subsets of 6 month primed donors. We found that antigen primed CD45R(low) CD4 T cells reverted (defaulted) with time to a CD45R(high) resting state, a change that was prevented by persisting antigen. The evidence suggests that CD4 memory T cells are partitioned into two different functional states (CD45R(high) and CD45R(low)) and that these determine the characteristics of the memory B cell response in terms of speed, size and longevity.     

Antigen-independent adhesion of CD45RA (naive) and CD45RO (memory) CD4 T cells to B cells.

We found that naive (CD45RA+) CD4 T cells have a lower capacity of adhesion to Epstein-Barr virus (EBV) immortalized B cells than memory (CD45RO+) CD4 T cells, as judged by conjugate formation. This would appear to be due to differences in the expression of adhesion molecules [lymphocyte function-associated antigen (LFA)-1, CD2]. However, kinetic studies showed that the degree of adhesion of naive T cells to B cells was stable over 60 min while that of memory T cells, like that of unseparated CD4 T cells, was characterized by a rapid formation and rapid dissociation of conjugates. This could be explained by a difference in the sensitivity of naive and memory CD4 T cells to down-regulation of antigen-independent adhesion by CD4-MHC class II interaction. Indeed, memory T cells also adhered stably to MHC class II(-) B cells. The adhesion of memory T cells, but not naive T cells, to MHC class II(+) B cells was sensitive to inhibition by OKT4a an anti-CD4 antibody, human immunodeficiency (HIV) gp160 (env) protein and a 12-mer peptide encompassing the 35-46 sequence of the HLA, DR beta 1 domain and previously shown to inhibit activation of HLA class II-restricted CD4 T cell responses. Since MHC class II expression did not influence the degree of conjugate formation by naive or memory CD4 T cells with B cells, CD4-MHC class II interaction does not appear to be involved in binding itself, but may down-regulate the adhesion of memory but not naive CD4 T cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

A second signal for T cell mitogenesis provided by monoclonal antibodies CD45 (T200)

The induction of T cell mitogenesis through CD3 is a complex process that requires at least two signals. The first one can be provided by Sepharose-bound CD3. The second one is normally provided by monocytes. The signal provided by Sepharose-bound CD3 is unable by itself to induce mitogenesis in monocyte highly depleted cells (MHDC). We describe here that the monoclonal antibody (mAb) 72-5D3 belonging to CD45 (T200), which was not mitogenic by itself, could replace monocytes when MHDC were activated by Sepharose-bound CD3. That is to say, in the absence of monocytes, mAb 72-5D3 gave a second signal necessary for T cell proliferation. Using eleven anti-CD45 mAb from other investigators we show that this effect is not a peculiar characteristic of 72-5D3 mAb. The effect of the mAb 72-5D3 was only effective in CD4-positive cells and was not observed when MHDC were activated with either soluble CD3 or concanavalin A. As both phorbol myristate acetate and mAb 72-5D3 can replace monocytes, a comparative study of their effects was undertaken. Phorbol myristate acetate but not mAb 72-5D3 induced proliferation of MHDC when recombinant interleukin 2 (rIL2) was added. On the other hand mAb 72-5D3 induced IL2 production in MHDC activated by Sepharose-bound CD3 and increased the IL2 production in Sepharose-bound CD3-activated peripheral blood mononuclear cells. In conclusion, data presented in this report indicate that the T200 molecule could be involved in T cell proliferation by giving a signal that induces the production of IL2 and bypasses the necessity of monocytes.     

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.     

Hyporesponsiveness of "naive" (CD45RA+) human T cells to multiple receptor-mediated stimuli but augmentation of responses by co-stimuli

Much remains to be clarified the functional capacities of the two major reciprocal subsets of human CD4+ cells which we interpret to be naive and memory cells. CD4+ naive (CD45RA+, LFA-3-) and memory (CD45R0+, LFA-3+) cells were rigorously purified by immunomagnetic negative selection. Their proliferation was measured in response to four protocols of receptor-mediated activation: soluble anti-CD3 mAb, plastic-immobilized anti-CD3 mAb, activating pairs of anti-CD2 mAb, and "superantigens" staphyloccocal enterotoxins A and B (SEA and SEB). Naive cells proliferated much less than memory cells to each of these four regimens although their capacity to respond was demonstrated by strong PHA-induced proliferation. Although three of the regimens depend on autologous monocytes, poorer naive cell responses are also observed to anti-CD3 mAb immobilized on plastic in the absence of monocytes; this implies an intrinsic hyporesponsiveness of naive cells, independent of their potentially weaker interaction with monocytes. Naive cells proliferated less than memory cells to superantigens SEA and SEB over a wide dose range; this assumes particular importance because such superantigens are believed to more closely mimic antigen-specific stimulation than anti-CD3 mAb. The possibility was explored that hyporesponsiveness of naive cells reflects the fact that naive cells require additional co-stimuli to facilitate their activation. In support of this concept, we observed that proliferation of naive cells to anti-CD3 mAb and SEA or SEB (but not to anti-CD2 mAb pairs) was consistently enhanced by pre-activation of monocytes present in the culture. Naive cell proliferative responses were augmented further in cultures supplemented with interleukin (IL) 1 beta and IL 6 or exposed to the co-stimulating mAb anti-CD28 and anti-CD44. The pattern of augmentation was dependent on the specific triggering regimen: anti-CD44 mAb was particularly effective in augmenting the response to superantigens, anti-CD28 mAb for the anti-CD3 response and IL 1 beta/IL 6 for that induced by anti-CD2 mAb pairs. With particular combinations of stimulus/co-stimuli naive cell proliferation was as strong as that of memory cells. We interpret these findings to indicate that naive cells are capable of responding to antigen, but that such responses are critically dependent on the available co-stimuli in vivo.     

Studies of lymphocyte transendothelial migration: analysis of migrated cell phenotypes with regard to CD31 (PECAM-1), CD45RA and CD45RO.

CD31 is a 130,000 MW cell-surface glycoprotein expressed on endothelial cells, polymorphonuclear leucocytes, monocytes and about 50% of peripheral blood lymphocytes, and it has been proposed that it plays a role in transendothelial migration. If it is involved in endothelial transmigration of lymphocytes then the proportion of CD31+ cells should be increased in the lymphocyte population which has crossed an endothelial monolayer. This was tested using two endothelial types, namely human umbilical vein endothelial cells (HUVEC) and rat high endothelial venule (RHEV) cells. As a control, lymphocyte CD45RA and CD45RO expression was also determined since there is a correlation between lymphocytes bearing these isoforms and different migratory patterns. Double labelling techniques showed a close correlation between CD31 and CD45RA expression. With HUVEC monolayers, the transmigrated lymphocyte population was depleted of CD31+ cells. This depletion was even more marked if the HUVEC monolayers had been stimulated with interleukin-1 beta (IL-1 beta). The migrated lymphocytes were enriched for CD31-CD45RO+ cells but depleted of CD31+CD45RA+ cells. In addition, lymphocyte populations depleted of CD31+ cells by immunopanning were also able to migrate across HUVEC monolayers. Taken together these data suggest that lymphocyte CD31 expression is not necessary for transmigration across HUVEC monolayers and, if anything, is negatively correlated with transmigration. With the second endothelial cell type, RHEV cells, there was no consistent change in the proportion of CD31+ lymphocyte in the transmigrated population, suggesting neither a positive nor a negative correlation between CD31+ expression and lymphocyte transmigration across RHEV cells. However, with both endothelial cell types, the migrated lymphocyte populations were enriched for the marker CD45RO. In conclusion, lymphocyte surface expression of CD31 is not necessary for transmigration across the endothelial cell types used in this study, but with both cell types an enrichment of CD45RO+ lymphocytes is seen in the migrated population.     

gamma(c) cytokines provide multiple homeostatic signals to naive CD4(+) T cells

Cytokines signaling through receptors sharing the common gamma chain (gamma(c)), including IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, are critical for the generation and peripheral homeostasis of B, T and NK cells. To identify unique or redundant roles for gamma(c) cytokines in naive CD4(+) T cells, we compared monoclonal populations of CD4(+) T cells from TCR-Tg mice that were gamma(c) (+), gamma(c) (-), CD127(-/-) or CD122(-/-). We found that gamma(c) (-) naive CD4(+) T cells failed to accumulate in the peripheral lymphoid organs and the few remaining cells were characterized by small size, decreased expression of MHC class I and enhanced apoptosis. By over-expressing human Bcl-2, peripheral naive CD4(+) T cells that lack gamma(c) could be rescued. Bcl-2(+) gamma(c) (-) CD4(+) T cells demonstrated enhanced survival characteristics in vivo and in vitro, and could proliferate normally in vitro in response to antigen. Nevertheless, Bcl-2(+) gamma(c) (-) CD4(+) T cells remained small in size, and this phenotype was not corrected by enforced expression of an activated protein kinase B. We conclude that gamma(c) cytokines (primarily but not exclusively IL-7) provide Bcl-2-dependent as well as Bcl-2-independent signals to maintain the phenotype and homeostasis of the peripheral naive CD4(+) T cell pool.     

Correlation between recent thymic emigrants and CD31+ (PECAM-1) CD4+ T cells in normal individuals during aging and in lymphopenic children

CD31(+)CD45RA(+)RO(-) lymphocytes contain high numbers of T cell receptor circle (TREC)-bearing T cells; however, the correlation between CD31(+)CD4(+) lymphocytes and TREC during aging and under lymphopenic conditions has not yet been sufficiently investigated. We analyzed TREC, telomere length and telomerase activity within sorted CD31(+) and CD31(-) CD4(+) lymphocytes in healthy individuals from birth to old age. Sorted CD31(+)CD45RA(+)RO(-) naive CD4(+) lymphocytes contained high TREC numbers, whereas CD31(+)CD45RA(-)RO(+) cells (comprising < or =5% of CD4(+) cells during aging) did not contain TREC. CD31(+) overall CD4(+) cells remained TREC rich despite an age-related tenfold reduction from neonatal (100 : 1000) to old age (10 : 1000). Besides a high TREC content, CD31(+)CD45RA(+)RO(-)CD4(+) cells exhibited significantly longer telomeres and higher telomerase activity than CD31(-)CD45RA(+)RO(-)CD4(+) cells, suggesting that CD31(+)CD45RA(+)RO(-)CD4(+) cells represent a distinct population of naive T cells with particularly low replicative history. To analyze the value of CD31 in lymphopenic conditions, we investigated six children after allogeneic hematopoietic stem cell transplantation (HSCT). Reemerging overall CD4(+) as well as naive CD45RA(+)RO(-)CD4(+) cells predominantly expressed CD31 and correlated well with the recurrence of TREC 5-12 months after HSCT. Irrespective of limitations in the elderly, CD31 is an appropriate marker to monitor TREC-rich lymphocytes essentially in lymphopenic children after HSCT.     

Role of B70/B7-2 in CD4+ T-cell immune responses induced by dendritic cells.

Dendritic cells (DC) are potent antigen-presenting cells (APC). However, the molecular basis underlying this activity remains incompletely understood. To address this question, we generated murine monoclonal antibodies (mAb) against human peripheral blood-derived DC. One such antibody, designated IT209, stained differentiated DC and adherent monocytes, but failed to stain freshly isolated peripheral blood mononuclear cells (PBMC). The antigen recognized by IT209 was identified as B70 (B7-2; also recently identified as CD86). Using this mAb we studied the role of B70 in CD4+ T-cell activation by DC in vitro. IT209 partly inhibited the proliferative response of CD4+ T cells to allogeneic DC and to recall antigens, such as tetanus toxoid (TT) and purified protein derivative (PPD) of tuberculin, presented by autologous DC. More importantly, the mAb had a potent inhibitory effect on the primary response of CD4+ T cells to autologous DC pulsed with human immunodeficiency virus (HIV) gp160 or keyhole limpet haemocyanin (KLH). Adherent monocytes, despite their expression of B70, failed to induce T-cell responses to these antigens. IT209-mediated inhibition of CD4+ T-cell responses was equivalent to that produced by anti-CD25 mAb, whereas an anti-CD80 mAb was only marginally inhibitory and did not augment the effect of IT209. These findings indicate that the B70 antigen plays an important role in DC-dependent CD4+ T-cell activation, particularly in the induction of primary CD4+ T-cell responses to soluble antigens. However, since activated monocytes, despite their expression of B70, failed to prime naive T cells to these antigens, our results suggest that additional molecules contribute to the functions of DC in CD4+ T-cell activation.     

Dynamic imaging of chemokine-dependent CD8+ T cell help for CD8+ T cell responses

Naive T lymphocytes move efficiently in lymphoid tissues while scanning dendritic cells in search of cognate complexes of peptide in major histocompatibility molecules. However, T cell migration ceases after recognition of cognate antigen. We show here that during the initiation of antigen-specific CD8(+) T cell responses, naive CD8(+) polyclonal T cells 'preferentially' interacted in an antigen-independent way with mature dendritic cells competent to present antigen to antigen-specific CD8(+) T cells. These antigen-independent interactions required expression of the chemokine receptor CCR5 on polyclonal T cells and increased the efficiency of the induction of naive, low-precursor-frequency CD8(+) T cell responses. Thus, antigen-specific CD8(+) T cells favor the priming of naive CD8(+) T cells by promoting the CCR5-dependent recruitment of polyclonal CD8(+) T cells to mature dendritic cells.     

Isoform-specific associations of CD45 with accessory molecules in human T lymphocytes.

Association of CD45 with surface molecules was investigated in human T lymphocytes by co-capping. CD45 appeared to be associated with the CD3/T cell receptor complex and with CD4 or CD8 molecules in memory, but not in naive T cells, as previously reported in the mouse. Associations of CD45 isoforms with accessory molecules were then identified with seven anti-CD45R monoclonal antibodies (mAb). An isoform-specific association pattern was observed: CD2 co-capped with CD45 molecules recognized by UCHL1 mAb (CD45R0). LFA-1 with molecules bound by 2H4 mAb (CD45RA), and both CD4 and CD8 with molecules reacting with MCA.347 mAb (whose isoform specificity was not known). Further information on the CD45 isoform(s) associated to CD4 and CD8 was sought by assessing the isoform specificity of MCA.347. Cross-competition experiments showed that it reacts with an epitope clearly different from those recognized by 2H4 and UCHL1, and only partially overlapping the PD7/26 epitope (CD45RB). Moreover, the competition between MCA.347 and PD7/26 was maximal in naive T cells and minimal both in memory T cells and in a subset expressing CD11b, a marker of granular lymphocytes. Immunoprecipitation experiments showed that MCA.347 binds to CD45 molecules with a molecular mass of 220, 205 and 190 kDa, the 190-kDa molecules not being recognized by 2H4, PD7/26 or UCHL1. These data indicate that MCA.347 recognizes amino acid sequences different from those coded by the exon A or B of the gene, and not expressed by CD45R0, suggesting that it binds to sequences coded by the exon C. In conclusion, this work shows that in human T cells different CD45 isoforms are associated to different surface molecules: LFA-1 is associated to CD45RA, CD2 to CD45R0 and CD4 and CD8 presumably to CD45RC. This peculiar behavior of CD45 suggests that it may play a crucial role in lymphocyte activation, probably by modulating the signals delivered to the cell by different receptor systems.