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CD26, a 110-kDa cell surface glycoprotein, exhibits dipeptidyl peptidase IV enzyme activity and plays an important role in T cell costimulation. In the present study, we examined both the exact adenosine deaminase (ADA) binding domain on CD26 and the functional consequences of mutated CD26 transfectants that were deficient for cell surface ADA. Using CD26 deletion, human-rat swap, and point mutations, we found that the residues of L340, V341, A342, and R343 on the CD26 molecule were essential amino acids for ADA binding. When these amino acids were mutated and transfected into Jurkat cells, the resultant CD26 transfectants expressed only CD26, not ADA, on the cell surface. The amount of IL-2 produced by wild-type and mutated CD26 transfectants was almost the same following stimulation with anti-CD3 plus PMA. However, the mutated CD26 transfectants were much more sensitive to the inhibitory effect of adenosine on IL-2 production than were the wild CD26 transfectants. These data suggest that ADA on the cell surface does not directly involve T cell activation. Conversely, CD26 alone does not result in modulating the inhibitory effect of adenosine. Only the ADA bound to CD26 on the cell surface was functional and could counteract the inhibitory effect of elevated extracellular adenosine.

CD40, a TNF receptor family member, plays a central role in T cell-mediated B cell activation. We have recently demonstrated that CD27, another TNF receptor family member, was also involved in B cell regulation and enhanced Ig production. In this report we compare CD27 and CD40 signals in B cell function. We selectively mimicked the effect of T cell help by addition to peripheral blood B cells activated with Staphylococcus aureus Cowan I strain and IL-2 of irradiated 300-19 cells transfected with either the CD70 (CD27 ligand) gene or the CD154 (CD40 ligand) gene, the vector alone, or both CD70 and CD154 genes. CD27 ligation induced only a slight increase in B cell proliferation compared with the dramatic enhancement induced by CD40 ligation; double ligation proved to be less efficient than CD40 ligation alone. In contrast, IgG production was increased only by CD27 ligation alone. Moreover, the CD27 signal was more efficient when it was given on day 2 of the culture rather than on day 0. Phenotypic analysis of the activated cells showed that CD27 ligation increased the percentage of cells showing a plasma cell profile (CD19-, CD38+), whereas upon CD40 ligation most of the cells still had a germinal center-like phenotype (CD19+, CD38+). Our results suggest that the CD27 and CD40 signals are not synergistic but, rather, are complementary and involve distinct steps of T cell-dependent B cell activation. CD27 may be more important in the induction of plasma cell differentiation at a time when the expansion phase has already occurred.

Studies toward the biologic and molecular understanding of programmed cell death have been stimulated by the recent identification of genes and their products that regulate apoptosis. A panel of mAbs has been raised against dying cells in the present study by immunizing mice with apoptotic Jurkat cells. One of these Abs, anti-7A6, was found to react with apoptotic cells. Using ELISA or flow cytometry, little reactivity of anti-7A6 was observed in normal or digitonin-permeabilized human peripheral blood lymphocytes and a number of hemopoietic cell lines tested. The Ab, however, strongly reacted with these cells when they were induced to undergo apoptosis by irradiation or treatment with apoptosis-inducing agents. Cell sorting and DNA fragmentation experiments revealed that 7A6-positive cells, but not 7A6-negative cells, had apparent DNA fragments characteristic of cells undergoing apoptosis. By immunoblot, under reducing conditions, anti-7A6 detected a 38-kDa protein band in the cell lysates prepared from apoptotic cells. Immunoelectron microscopy showed the 7A6 Ag to be localized to the membrane of mitochondria in apoptotic Jurkat cells. These results indicate that anti-7A6 defines a novel epitope on the mitochondrial membrane protein that appears to be exposed on cells undergoing apoptosis, suggesting that the 7A6 molecule may be involved in the molecular cascade of apoptotic cell death.

Our previous studies demonstrated that upon activation, monocytes (Mo) were able to sensitize peripheral blood T (PBT) cells to apoptosis induced by treatment with PMA. However, it is unknown what gene products provide the death signal to the sensitized PBT cells and how activated Mo enable PBT cells to become susceptible to apoptosis. Here, we show that PBT cells, but not Mo, express functional Fas ligand upon treatment with PMA. Moreover, this Mo-dependent T cell apoptosis could be blocked by a Fas-Ig fusion protein, as well as by a nonlytic mAb against Fas molecule. These results strongly suggest involvement of Fas-Fas ligand interaction in the death of PBT cells. Unlike Fas-induced apoptosis, however, Mo-dependent T cell death was completely inhibited by overexpression of the Bcl-2 protein, and PMA alone was sufficient to trigger apoptosis in T cells when Mo were included in culture. Furthermore, anti-CD11a, anti-CD18, or anti-CD45/CD45RA mAbs; could prevent PBT cells from death triggered by PMA plus Mo, suggesting that these Ags participate in the apoptotic process. The participation of CD45RA in the death of PBT cells was further demonstrated by the observation that the J45.01 cell line, a CD45-deficient variant of Jurkat cells, did not undergo apoptosis by this Mo-dependent mechanism. When transfected with cDNA encoding CD45RA, J45.01 cells acquired apoptotic response to PMA stimulation in the presence of Mo to a similar, but lesser, degree than normal Jurkat cells.

This study demonstrates that the 175-kDa form of dipeptidyl peptidase IV (DPPIV) found in normal human serum is identical with a similarly-sized Ag, DPPT-L, found to be rapidly expressed on the surface of activated T cells. As activation progresses, the expression of DPPT-L reaches a peak on day 3, after which expression falls, whereas expression of the 105-kDa CD26/DPPIV detected by the mAb 1F7 increases, as does the ability to bind adenosine deaminase. The loss of DPPT-L from the surface of activated T cells correlates exactly with the appearance of DPPT-L and DPPIV activity in serum-free tissue culture medium. The release of DPPIV was generally greater from CD4+ cells than from CD8+ T cells, and within the CD4+ subset, the CD45RO+ subset was the major source, which correlated with surface expression before culture. We show that the DPPIV released from activated T cells is antigenically, biochemically, and enzymatically similar to DPPIV circulating in the serum and is distinct from the DPPIV activity of 105-kDa CD26. The T cell-released DPPIV is able to function as a costimulating molecule for the response to the recall Ag, tetanus toxoid, at levels similar to those at which recombinant soluble CD26 and serum DPPIV exhibit costimulatory function, suggesting that the released DPPIV may serve an important immunoregulatory function in vivo, both locally and within the systemic circulation.

CD26, a T cell activation Ag, also known as dipeptidyl peptidase IV, is directly associated with adenosine deaminase (ADA) on the surface of T cells and T cell lines. In the present study, we examined both the binding of ADA and CD26 and the functional consequences of this interaction. We found that ADA was associated with CD26 on T cell lines lacking either ADA or dipeptidyl peptidase IV enzymatic activity, indicating that the association between dipeptidyl peptidase IV and ADA did not require enzymatic activity. Moreover, using immunoelectron microscopy, we demonstrated that CD26 and ADA co-localized on the cell surface, but not inside cells, suggesting that CD26 did not transport ADA to the surface. In keeping with this observation, we showed that human CD26-transfected murine pre-B cell lines lacking human ADA acquired ADA from an extracellular source. More importantly, adenosine in the absence of cell surface ADA inhibited T cell proliferation and IL-2 production induced by various stimuli. On the other hand, cells expressing ADA and CD26 on the surface were much more resistant to the inhibitory effect of adenosine. These data suggest that ADA on the cell surface is involved in an important immunoregulatory mechanism by which released ADA binds to cell surface CD26, and this complex is capable of reducing the local concentration of adenosine.

The engagement of CD27 with its ligand CD70 is considered to play an important role in T cell costimulation. In the present study, we investigated both the kinetics of CD70 expression and the contribution of its interaction with CD27 in T cell immune responses. CD70 was found to be expressed almost equally on both activated CD4 and CD8 T cells. On subsets of CD4 T cells, however, CD70 expression was induced preferentially on the CD45R0 T cell population after activation, whereas its expression was not noted on CD45RA T cells for almost 2 wk following activation. In long-term culture with media containing T cell growth factor (TCGF) and rIL-2, the expression of CD70 was increased markedly on CD45R0 T cells and minimally expressed on CD45RA T cells. In addition, strong surface expression of CD70 was observed on T cell clones originally derived from CD45R0+ CD4 T cells, whereas T cell clones originally derived from CD45RA+ CD4 T cells showed lower levels of expression. The addition of irradiated, activated CD45R0 T cells to CD45RA T cells caused a down-regulation of CD27 expression and an up-regulation of CD25 expression. These changes were blocked by addition of the anti-CD70 mAb, suggesting that direct contact between CD45R0 T cells and CD45RA T cells via CD27/CD70 occurred, leading to the activation of CD45RA T cells as measured by CD25 expression. These observations strongly support the notion that the engagement of CD27 plays an important regulatory role in the communication of subsets of CD45R0 and CD45RA T cells.

CD27 is a 120-kDa transmembrane homodimeric molecule expressed on the majority of T cells, B cells, and NK cells that belongs to the TNFR/nerve growth factor receptor family. The interaction between CD27 and its ligand, CD70, is thought to play an important role in T cell activation. In this paper we have examined the signal-transducing potential of CD27 in T cell costimulation. Anti-CD27 mAb, anti-1A4, induced substantial proliferation of peripheral blood T cells in the presence of a suboptimal dose of PMA, phytohemagglutinin, anti-CD2, or anti-CD3 together with a second Ab to cross-link the CD27 molecule. This T cell proliferation was also observed by using CD70 transfectant cells. CD27 cross-linking maximally induced proliferation of CD45RA+CD4 T cells but only slightly induced proliferation of CD45RO+CD4 T cells. CD27-mediated T cell proliferation did not seem to be dependent on the IL-2/IL-2R system because no detectable level of IL-2 was secreted, and only a partial inhibition was observed with anti-IL-2 and anti-IL-2R Abs. Furthermore, an increase in intracellular Ca2+ was observed in PMA-treated T cells when the CD27 molecule was cross-linked. More importantly, CD27 ligation induced protein tyrosine phosphorylation, especially 70 kDa of cellular substrate, including ZAP-70, in T cells. Herbimycin A, a protein tyrosine kinase inhibitor, and staurosporine, a protein kinase C inhibitor, blocked T cell proliferation induced by CD27 ligation, suggesting the possibility that the activation of protein tyrosine kinase and protein kinase C is required for CD27-mediated T cell costimulation. These results clearly demonstrate that the CD27/CD70 interaction induces costimulatory signals in T cells, especially CD45RA+ naive T cells, indicating that CD27 serves as a T cell signal-transducing molecule.

CD27 is a disulfide-linked 120-kDa transmembrane glycoprotein expressed on the majority of T cells, B cells, and NK cells; it has homology to a family of molecules that includes the receptors for nerve growth factor and TNF. Previous studies strongly suggest that the CD27 molecule plays a key role in the process of T cell activation. To further determine its role in T cell activation, a recombinant soluble molecule composing only the extracellular domain of CD27 was produced by transfection of Chinese hamster ovary cells. We have defined the binding properties of recombinant soluble CD27 (rsCD27) to CD27 ligand (CD27L) cDNA transfected NIH 3T3 cells and have determined its functional effects on in vitro T cell activation as well as on PWM-driven B cell IgG synthesis. rsCD27 bound specifically to CD27L and the binding was inhibited by one of our anti-CD27 mAbs, anti-1A4, suggesting that the 1A4 epitope of CD27 plays a role in the binding to CD27L. Functionally, rsCD27 inhibited T cell proliferation induced by various stimuli, such as PHA, tetanus toxoid, and anti-CD2, as well as PWM-driven B cell IgG synthesis, similar to the effects of adding anti-1A4. Determination of CD27L expression showed that CD27L mRNA is induced rapidly on activated T and B cells. Taken together, these results provide direct evidence that CD27-CD27L interaction plays a critical role in T cell activation as well as in T cell-dependent B cell IgG synthesis, suggesting that the CD27-CD27L interaction may constitute a component of the T cell-T cell or T cell-B cell interaction seen after activation with Ag or mitogen.

Antibodies to the CD6 Ag have been described as having pan-T cell reactivity. We have recently demonstrated, however, that after treatment of PBL with an anti-CD6-blocked ricin-conjugated immunotoxin, clonal populations of CD3+, CD6- cells can be identified. Herein we show that through dual parameter staining of freshly isolated E-rosette+ cells, an average of 5 to 6% of either CD3+ or CD5+ cells express little or no CD6 on their surface. After negative selection by antibody-coated paramagnetic bead depletion, expanded CD6- T cells were shown to be CD1a-, CD2+, CD3+, CD5+, CD16-, CD56-, TCR-gamma/delta-, and consisted of both CD4+ and CD8+ cells. Furthermore, staining of digitonin permeabilized cells showed no cytoplasmic expression of the CD6 Ag and CD6 mRNA was not detected by Northern blot analysis. Identical staining patterns were observed for T cell clones isolated through bead depletion or immunotoxin treatment and expanded with either PHA or immobilized anti-CD3 mAb. It was also found that, relative to unfractionated T cells, the surface expression of CD5 was significantly diminished on CD6- T cells. Functionally, freshly isolated CD6- T cells showed substantially reduced alloreactivity in MLR compared with unfractionated E-rosette+ cells, yet both gave similar proliferative responses to either PHA or soluble tetanus toxin Ag. We conclude that there exists a minor subpopulation of mature T cells in peripheral blood that lack CD6. The diminished alloreactivity of these cells may help to explain the low incidence of graft-vs-host disease, despite high levels of engraftment, that has been reported in allogeneic bone marrow transplant patients receiving marrow treated with anti-CD6 (T12) mAb plus C'.

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.

The CD27 Ag is expressed by the majority of resting T lymphocytes and appears to play a crucial role in T cell activation. We found that some resting peripheral blood NK cells also express CD27. Furthermore, CD27 expression was up-regulated on NK cells stimulated by IL-2. The cytolytic activity of IL-2-activated, but not resting, NK cells was inhibited by an anti-CD27 mAb (anti-1A4). However, anti-1A4 did not affect conjugate formation between IL-2-activated NK cells and tumor cell targets. In contrast, anti-1A4 inhibited CD2-mediated calcium mobilization and the serine esterase activity of NK cell granules. These inhibitory effects could be mediated in part by increase in intracellular cAMP levels induced by anti-1A4. Our results suggest that the CD27 Ag plays an important role in the regulation of activated NK cells.

A cDNA encoding the T cell activation Ag CD26 was isolated from human PHA-activated T cells by using an expression cloning method. The nucleotide sequence obtained predicts a protein of 766 amino acids of type II membrane topology, with six amino acids in the cytoplasmic region. The predicted amino acid sequence of the Ag was 85% homologous to that of the dipeptidyl peptidase IV enzyme isolated from rat liver. Derivatives of the human leukemic T cell line Jurkat transfected with a CD26 expression plasmid were established. Characterization of the CD26 Ag expressed by the transfected Jurkat cells revealed that the Ag could be immunoprecipitated as a 110-kDa molecule similar to that found on peripheral blood T cells and that the Ag had dipeptidyl peptidase IV activity. Functional analysis of these Jurkat transfectants showed that cross-linking of the CD26 and CD3 Ag with their respective antibodies resulted in enhanced intracellular calcium mobilization and IL-2 production. These results provide direct evidence that the CD26 Ag plays a role in T cell 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.

The TCR complex is composed of a clonotypic heterodimer (Ti alpha:beta or gamma:delta) noncovalently associated with the CD3 complex (gamma, delta, and epsilon), and with one or more disulfide-linked dimers whose components are designated zeta and eta. zeta and eta are alternative transcripts of a common gene and are structurally related to the gamma-subunit of the FcR for IgE expressed on mast cells and basophils (Fc epsilon RI). Recent evidence suggests that gamma can also be expressed in natural killer cells and in a murine cytotoxic T cell line, CTLL. Because zeta, eta, and gamma have the potential to join together to form disulfide linked homo- and heterodimers, it has been postulated that alternative dimeric forms composed of these zeta-related subunits might subserve unique signal transducing functions in hematopoietic cells. We have used mAb reactive with zeta and gamma to systematically examine the expression of these zeta-related dimers in human T cells, NK cells, and thymocytes. Our results show that each cell type expresses characteristic combinations of zeta-related homo- and hetero-dimers, and are therefore consistent with the possibility that these subunits contribute to the functional heterogeneity of lymphocyte subsets.

We have recently shown that solid-phase immobilization of anti-1F7 recognizing the 110-kDa CD26 Ag is comitogenic for human peripheral blood T cell activation via both the CD3 and CD2 pathways. We have also demonstrated that binding of anti-1F7 leads to the disappearance of CD26 surface expression, and this anti-1F7-induced modulation results in an increase in anti-CD3 or anti-CD2-mediated peripheral blood T cell activation. In this report, we extended these findings by examining the expression and functional relationship of 1F7 on the CD3 and CD2 pathways of activation of human thymocytes. We now demonstrated that most of the anti-1F7 reactivity is found on medullary thymocytes, the population of thymocytes expressing high level of CD3 (CD3H). We have also shown that binding of anti-1F7 can induce a decrease in CD26 surface expression, with no detectable effect on the surface expression of CD3 or CD2. Most importantly, we showed that solid-phase immobilization of anti-1F7 has a comitogenic effect on thymocyte activation induced by anti-CD3 but not anti-CD2. In addition, anti-1F7-induced modulation of CD26 results in an enhancement in CD3-mediated but not CD2-mediated human thymocyte activation. The observed functional effect of CD26 on the CD3/TCR pathway of activation is mainly restricted to mature thymocytes as distinguished by high surface expression of CD5, although CD26 is also functionally associated with the CD3/TCR pathway on cells expressing low level of CD5. Demonstrating that CD26 involvement in the regulation of human thymocyte activation is restricted mainly to the CD3 pathway, unlike its involvement with both the CD3 and CD2 pathways of mature peripheral blood T lymphocyte activation, our data hence suggested that CD26 may play a role in thymic differentiation and maturation via the differential engagement of the CD3 pathway.

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.

The CD45 Ag family is comprised of at least five isoforms generated by the alternative splicing of three exons encoded by a single leukocyte common Ag gene. These isoforms, differentially expressed on subsets of T cells, are widely believed to delineate maturational status. Thus, cells expressing the high molecular mass CD45RA+ isoforms (220 and 205 kDa) are believed to represent naive cells, whereas those lacking CD45RA and expressing high density CD45RO (180 kDa) represent memory cells. This is based on findings that after activation, CD45RA+ cells "convert" to the CD45RA- phenotype, losing CD45RA and gaining CD45RO. This conversion is believed to be both unidirectional and irreversible. We now report a strategy for the development of polyclonal CD45RA+ cell lines derived from peripheral blood CD4+CD45RA+ cells. However, in such cell lines, CD45RA expression was not constant, but varied cyclically with restimulation. After restimulation, CD45RA expression decreased significantly, nadiring on days 5 to 7. However, CD45RA was fully re-expressed by days 14 to 16. These cells coexpressed high density CD45RO, which did not vary through the stimulatory cycle. Cells sorted CD45RA- on day 7 of the cycle rapidly reexpress CD45RA. Metabolic labeling revealed that the 220 kDa CD45RA isoform is no longer synthesized. Synthesis of the 205 kDa (CD45RA) isoform decreased significantly early after stimulation, but then increased back to baseline after day 12. The 180-kDa (CD45RO) isoform followed an opposite pattern. Our results document that alternative splicing of the different CD45 isoforms is highly regulated after activation and that conversion from CD45RA+ to CD45RA- is neither unidirectional, nor irreversible.

Fibronectin synergized with anti-CD3 antibody to promote CD4 cell proliferation in a serum-free culture system whereas no proliferation was observed when CD4 cells were cultured with anti-CD3 alone or fibronectin alone. In addition, anti-CD29 (integrin beta 1) as well as anti-VLA-5 (human fibronectin receptor) antibodies blocked this CD4 cell activation in this system. Although anti-CD3 alone or fibronectin alone cannot induce IL-2 message by CD4 cells, the combination of anti-CD3 plus fibronectin induced IL-2 message by CD4 cells. In an analysis of the molecular mechanism by which IL-2 message was generated, we showed that a fibronectin-VLA-5 fibronectin receptor interaction may contribute an independent signal distinct from the CD3 pathway of activation by the induction of an AP-1 transcriptional factor. Thus the VLA-5 fibronectin receptor on CD4 cells can play a complementary role in CD3-TCR-mediated signal transduction through its interaction with fibronectin.

NK cells are large granular lymphocytes capable of killing certain tumor cells and virally infected cells in a non-MHC-restricted manner. NK cells can also effect an antibody dependent cytotoxicity that is triggered by CD16, an FcR for IgG. In NK cells, CD16 is expressed in association with zeta, a signal transducing subunit of the TCR complex. Here we show that, just as T cell activation via the TCR complex results in tyrosine phosphorylation of zeta TCR, NK cell activation via CD16 results in tyrosine phosphorylation of zeta NK. Whereas antibody-dependent cytotoxicity also results in tyrosine phosphorylation of zeta, natural cytotoxicity does not. Our results indicate that zeta functions as a transducing element for antibody dependent, but not antibody independent killing by NK cells. Consequently, NK cells are likely to express at least two distinct receptor complexes capable of triggering cytolytic effector function.

In this paper, we examined in detail the ability of anti-1F7 to modulate 1F7 (CD26) surface expression as well as analyzed the functional relationship between the surface expression of CD3, CD2, and CD26 and human T cell activation. We showed that anti-1F7-induced modulation is an energy-dependent process that occurs via capping and internalization of the Ag-antibody complex. Although the recovery rate for Ag reexpression of 1F7 following optimal modulation is relatively delayed, reexpression of 1F7 is greatly accelerated following phorbol ester treatment. Most importantly, we demonstrated that modulation of the CD26 Ag leads to an enhancement in the proliferative activity of modulated human T cells treated with anti-CD3 or anti-CD2, which is preceded by an enhancement in Ca2+ mobilization. CD26 modulation also led to an increase in anti-CD3- or anti-CD2-mediated T cell clone proliferation. Finally, whereas modulation of the CD26 Ag has an effect on CD3- or CD2-induced T cell activation, modulation of the CD3/TCR complex inhibits the proliferative response of T cells incubated with anti-CD3 plus anti-1F7 or anti-CD2 plus anti-1F7. However, modulation of the CD2 structure does not affect anti-CD3- plus anti-1F7-induced human T cell activation. The above results thus provide additional evidence that the CD26 Ag plays an integral role in the regulation of human T cell activation.

We have recently developed a mAb, anti-1F7, which defines a family of structures found to include the molecule recognized by anti-Ta1 (CD26). In this paper, we demonstrated that binding of 1F7 by solid-phase immobilized anti-1F7 mAb but not anti-Ta1 mAb has a comitogenic effect by inducing proliferation of human CD4+ T lymphocytes in conjunction with submitogenic doses of anti-CD3 or anti-CD2. The proliferative response induced via the CD3-1F7 or CD2-1F7 pathways is associated with the IL-2 autocrine pathway, including IL-2 production. IL-2R expression and anti-IL-2R (Tac) inhibition. Furthermore, solid-phase immobilization of anti-1F7 but not anti-Ta1 acts in conjunction with submitogenic doses of PMA to mediate a comitogenic effect in the absence of anti-CD3 or anti-CD2, leading to CD4+ T cell proliferation. PMA treatment, in the meantime, leads to enhanced expression of 1F7 on the T cell surface. Despite its functional association with both pathways of activation, however, the 1F7 structure is not comodulated with the CD3/TCR complex nor the CD2 molecule. These findings thus suggest that the CD26 Ag is involved in CD3 and CD2-induced human CD4+ T cell activation.

Variations in intracellular Ca2+ levels in developing thymocytes are likely to play a major role in both the activation-associated differentiation of thymocytes and in the selection or clonal deletion of cells. Here we examine the role of CD4, CD8, CD2, and CD45 in the regulation of intracellular Ca2+ levels in mature and immature thymocytes. Mature and immature thymocytes, distinguished on the basis of their CD5 expression, were analyzed simultaneously for their ability to mobilize Ca2+ after coaggregation of their CD3/TCR with other thymic surface Ag. Flow cytometric analysis by using Indo-1 showed that coaggregation of CD4, CD8, and CD2 with CD3/TCR clearly enhances a minimal signal delivered via CD3/TCR on immature thymocytes. Coaggregation with class I MHC had no discernible effect. The responsiveness of immature thymocytes correlated strictly with CD3 surface expression, such that loss of responsiveness occurred with reduced CD3 cell-surface density. However, even thymocytes with very low CD3 expression were able to respond to triggering via CD3 under optimal conditions, indicating that the CD3 signal-transducing mechanism is functional on early thymic cells. Intracellular increases in Ca2+ concentrations induced via CD3, could effectively be inhibited by cross-linking of CD45 and CD3 on immature thymocytes. Although triggering via CD2 alone induced a strong Ca2+ flux, prolonged incubation with activating anti-CD2 antibodies made thymocytes refractory to subsequent triggering. Refractoriness was associated with partial loss of surface CD3 and CD3 zeta. Our results indicate that thymic surface Ag are differentially involved in the regulation of intracellular Ca2+ levels in immature as well as mature thymocytes.

We have recently described a novel method for the production and characterization of mAb reactive with T cell-restricted intracellular antigens. From a panel of antibodies that react specifically with permeabilized T lymphocytes but not with permeabilized B lymphocytes or native T cells, we have selected one, designated TIA-1, that reacts with 20 to 36% of digitonin permeabilized peripheral blood T lymphocytes. Flow cytometric analysis of purified CD4+ and CD8+ subsets showed TIA-1 to recognize a subpopulation of 49 to 64% of CD8+ lymphocytes. Little or no reactivity with CD4+ resting T lymphocytes was observed. TIA-1 did not react with any of a panel of T cell lines, B cell lines, or monocytoid cell lines. TIA-1 reacted strongly with NK cell clones and CD8+ cytolytic T cell clones, and less strongly with CD4+-activated T cell clones, suggesting a preferential expression in cells possessing cytolytic potential. Cell fractionation experiments showed TIA-1 to be membrane associated. Furthermore, Percoll gradient fractionation of a cytolytic T cell clone (T4T8C1) showed the majority of TIA-1 to be contained in a low density membrane fraction that also contained serine protease activity. Immunoelectron microscopy showed TIA-1 to decorate the membranes of electron lucent and electron dense cytoplasmic granules in this same cytolytic T cell clone. Biochemical analysis showed TIA-1 to be a 15-kDa protein in unstimulated T cells. Upon activation with Con A or anti-CD3 antibodies. TIA-1 was induced to form disulfide linked dimers, trimers, and tetramers of the basic 15-kDa unit. Taken together, our data suggest that TIA-1 is a cytolytic granule associated protein that may define a subpopulation of resting CD8+ T lymphocytes possessing cytolytic potential.

Attractin is a rapidly upregulated membrane-associated molecule on activated T cells. It is a member of the CUB family of extracellular guidance and development proteins, sharing with them a protease activity similar to that of Dipeptidyl peptidase IV (DPPIV/CD26). Most remarkably, and in sharp contrast to CD26, it is released from the T cell and is presumed to be a major source of a soluble serum-circulating attractin. Genomic sequencing reveals that the soluble form is not a proteolytic product of the membrane form, but is in fact the result of alternative splicing. Recent results prove that the loss of murine membrane attractin results in the mahogany mutation with severe repercussions upon skin pigmentation and control of energy metabolism. In each of these latter instances, there is a strong likelihood that attractin is moderating the interaction of cytokines with their respective receptors. We propose that attractin is performing a similar function in the immune system through capture and proteolytic modification of the N-terminals of several cytokines and chemokines. This regulatory activity allows cells to interact and form immunoregulatory clusters and subsequently aids in downregulating chemokine/cytokine activity once a response has been initiated. These two properties are likely to be affected by the balance of membrane-expressed to soluble attractin.

A breast tumor hypoxia model used to simulate conditions which may exist within an enlarging tumor was examined using documented methods for identifying mechanisms of cell death and compared to the mitochondrial membrane-specific APO2.7 antigen expression. Hypoxic conditions were induced by holding cell pellets of MDA-MB-175-VII breast carcinoma cells in tightly capped centrifuge tubes for up to 10 days. Cells were harvested at 1.5, 3, 4.5, 6, 12, 18, and 24 h, and each 24 h thereafter to 10 days. APO2.7 was monitored in unprocessed cells (no permeabilization prior to staining) for all time points and processed cells (permeabilized prior to staining) for only the first 24 h. Cell viability probes trypan blue and anti-tubulin antibody showed a rapid increase in staining over the first 24 h, as did the phosphatidylserine-specific annexin V and DNA fragmentation by flow cytometry (range of 60-81% positive staining). Light scatter changes indicative of cell death were also quite remarkable. APO2.7 staining never exceeded 42% of the cell pellet over the 10 days of testing compared to greater than 95% staining for all other methods tested. When APO2.7 antigen expression was examined with respect to depth in the cell pellet, it was apparent that cells deeper in the pellet expressed APO2.7 more rapidly; however, fewer cells stained and cells showed fewer apoptotic features on an ultrastructural level than cells at the cell media interface. The study indicates that the anti-APO2.7 antibody may be able to discern apoptotic and incomplete apoptotic cells from necrotic MDA-MB breast cancer cells, traversing a heterogeneous pathway to cell death induced by hypoxia.

To clarify the different anti-CD26 mAbs corresponding different functions of CD26, the correlation of the epitopes defined by anti-CD26 mAbs and the functions of CD26 have been studied. Using truncated, human-rat CD26 swap mutants and cross-blocking studies, 13 anti-CD26 mAbs were divided into 5 separate groups. These 5 epitopes were localized between the 1-247th, 248-358th, 359-449th (closer to the 359th amino acid), 450-577th and 359 653th amino acid regions. MAbs against two of these five epitopes, the 248-358th and 359-449th amino acid regions, were associated with inducing modulation of CD26 and T-cell costimulation through the CD3 pathway. Furthermore, mAbs against one of these epitopes, the 359-449th amino acid region, appeared to encompass the ADA binding domain. Analysing the avidity of each mAb to the CD26 molecule using DPPIV enzymatic activity as an indicator, we found that the function of CD26 had little correlation with the avidity of anti-CD26 mAbs, suggesting that distinct epitopes defined by anti-CD26 mAbs appeared to be associated with different functions of CD26. These results will be very useful in the further definition of the functional domains of CD26.

CD26, a 110-kDa cell surface glycoprotein, exhibits dipeptidyl peptidase IV enzyme activity and plays an important role in T cell costimulation. In the present study, we examined both the exact adenosine deaminase (ADA) binding domain on CD26 and the functional consequences of mutated CD26 transfectants that were deficient for cell surface ADA. Using CD26 deletion, human-rat swap, and point mutations, we found that the residues of L340, V341, A342, and R343 on the CD26 molecule were essential amino acids for ADA binding. When these amino acids were mutated and transfected into Jurkat cells, the resultant CD26 transfectants expressed only CD26, not ADA, on the cell surface. The amount of IL-2 produced by wild-type and mutated CD26 transfectants was almost the same following stimulation with anti-CD3 plus PMA. However, the mutated CD26 transfectants were much more sensitive to the inhibitory effect of adenosine on IL-2 production than were the wild CD26 transfectants. These data suggest that ADA on the cell surface does not directly involve T cell activation. Conversely, CD26 alone does not result in modulating the inhibitory effect of adenosine. Only the ADA bound to CD26 on the cell surface was functional and could counteract the inhibitory effect of elevated extracellular adenosine.

A recently described mitochondrial membrane protein-specific monoclonal antibody, APO2.7, was examined for monitoring early apoptotic responses in anti-CD95 (7C11)-induced Jurkat cells. Jurkat cells were harvested at 1.5, 3, 4.5, 6, 12, and 18 h after induction of apoptosis, and APO2.7 antibody monitored in unprocessed (no permeabilization agent used prior to staining) and processed (permeabilized prior to staining) cells. Light-scatter changes (decreased forward-scatter and increased side-scatter) by flow cytometry were observed after 3 h, and detection of cell permeability in unprocessed cells, as measured by light microscopic examination of Trypan blue-stained cells and flow cytometric detection of tubulin, showed little change until after 6 h. In addition, unprocessed cells stained with APO2.7 antibody showed little increase in staining until after 6 h following induction of apoptosis, when DNA fragmentation was demonstrated by flow cytometry and gel electrophoresis; however, processed cells stained with APO2.7 antibody showed significant increase in staining after 1.5 h. Detection, using annexin V and flow cytometry, of phospholipid membrane asymmetry from exposure of phosphatidylserine showed greater, apparent nonspecific staining in noninduced cells as compared to the other markers of apoptosis, but nearly paralleled the results of APO2.7 staining in processed cells from 3-18 h following CD95 induction of apoptosis. The data presented herein indicate that the mitochondrial membrane protein-specific antibody, APO2.7, is useful as a marker for the detection of apoptotic cells.

CD40, a TNF receptor family member, plays a central role in T cell-mediated B cell activation. We have recently demonstrated that CD27, another TNF receptor family member, was also involved in B cell regulation and enhanced Ig production. In this report we compare CD27 and CD40 signals in B cell function. We selectively mimicked the effect of T cell help by addition to peripheral blood B cells activated with Staphylococcus aureus Cowan I strain and IL-2 of irradiated 300-19 cells transfected with either the CD70 (CD27 ligand) gene or the CD154 (CD40 ligand) gene, the vector alone, or both CD70 and CD154 genes. CD27 ligation induced only a slight increase in B cell proliferation compared with the dramatic enhancement induced by CD40 ligation; double ligation proved to be less efficient than CD40 ligation alone. In contrast, IgG production was increased only by CD27 ligation alone. Moreover, the CD27 signal was more efficient when it was given on day 2 of the culture rather than on day 0. Phenotypic analysis of the activated cells showed that CD27 ligation increased the percentage of cells showing a plasma cell profile (CD19-, CD38+), whereas upon CD40 ligation most of the cells still had a germinal center-like phenotype (CD19+, CD38+). Our results suggest that the CD27 and CD40 signals are not synergistic but, rather, are complementary and involve distinct steps of T cell-dependent B cell activation. CD27 may be more important in the induction of plasma cell differentiation at a time when the expansion phase has already occurred.

Pentoxifylline (PTX), a methylxanthine derivative, was examined for its effects on T cell proliferation and cytokine production stimulated by cross-linking anti-CD3 alone, anti-CD3 with PMA, anti-CD3 with anti-CD26, or anti-CD3 with anti-CD28 mAb, respectively. PTX at a 3.5 x 10(-5) M concentration significantly inhibited T cell proliferation and the production of tumor necrosis factor-alpha, interleukin-2, and interleukin-4. Moreover, this effect was selective for stimulation by cross-linking anti-CD3 with PMA, or anti-CD3 with anti-CD26, but not by cross-linking anti-CD3 with anti-CD28. These results suggest that the inhibitory effect of PTX on T cell activation involves the CD3 and CD26, but not the CD28 signal pathway.

In the present study, we analyzed human renal allografts using immunohistochemical techniques to determine the site, identity, and frequency of (a) cytotoxic and apoptotic cells, as identified by staining for GMP-17 (TIA-1), a component of cytotoxic granules; and (b) DNA fragmentation in situ, as detected by the TUNEL method. In acute cellular rejection (n = 15), GMP-17+ mononuclear cells accounted for 29% +/- 12% of the infiltrating cells in the interstitium (341 +/- 164/mm2) and were significantly more concentrated in tubulitis lesions, where they amounted to 65% +/- 14% of the mononuclear cells (96 +/- 61/mm2) (p0.01 versus interstitium). GMP-17+ mononuclear cells were also found in sites of endothelialitis. An estimated 80% of the GMP-17+ lymphocytes expressed CD8, and 10% to 20% expressed either CD4 or the macrophage marker CD14. The latter finding led us to analyze normal peripheral blood monocytes by flow cytometry, all of which were found to contain GMP-17. NK cells and neutrophils, which are known to express GMP-17, were detected only rarely in allografts. Specimens with cyclosporine A toxicity (n = 7) or acute tubular necrosis (n = 13) showed fewer GMP-17+ cells in the interstitium (22 +/- 46/mm2 and 62 +/- 50/mm2, respectively) and tubules (2 +/- 6/mm2 and 10 +/- 10/mm2, respectively) (all p0.01 versus rejection). These differences were due largely to less intense mononuclear cell infiltration. In cyclosporine A toxicity, however, the percentages of GMP-17+ mononuclear cells within tubules and the interstitium were significantly lower than in rejection (p = 0.02), whereas in acute tubular necrosis significantly lower percentages were found in the tubules (p = 0.04) but not in the interstitium. Native kidneys with end-stage diabetic nephropathy (n = 5) had very low proportions of GMP-17+ cells in interstitial infiltrates (7% +/- 6%) and in tubules (11% +/- 15%), although the infiltrates were focally intense (517 +/- 355/mm2). TUNEL+ cells were found in acute cellular rejection, predominantly in areas with intense mononuclear infiltrates and also within lesions of tubulitis and endothelialitis. Although some TUNEL+ cells were intrinsic renal cells, most appeared to be infiltrating mononuclear cells, and we were able to detect CD3 in some. In areas of intense cellular infiltration, the percentages of TUNEL+ cells (range, 0.5% to 4.2%) were comparable to those seen in the rat thymus, indicating a high level of apoptosis. Overall, in the allograft samples, the numbers of GMP-17+ cells and TUNEL+ cells were significantly correlated (r = 0.79; p0.01). These data provide new evidence that T cell (and possibly macrophage)-mediated cytotoxicity plays an important role in acute renal allograft rejection, particularly in the case of tubular injury, and furthermore suggest that apoptosis may be a mechanism not only for graft cell destruction, but also for elimination of activated T cells in the infiltrate.

Studies toward the biologic and molecular understanding of programmed cell death have been stimulated by the recent identification of genes and their products that regulate apoptosis. A panel of mAbs has been raised against dying cells in the present study by immunizing mice with apoptotic Jurkat cells. One of these Abs, anti-7A6, was found to react with apoptotic cells. Using ELISA or flow cytometry, little reactivity of anti-7A6 was observed in normal or digitonin-permeabilized human peripheral blood lymphocytes and a number of hemopoietic cell lines tested. The Ab, however, strongly reacted with these cells when they were induced to undergo apoptosis by irradiation or treatment with apoptosis-inducing agents. Cell sorting and DNA fragmentation experiments revealed that 7A6-positive cells, but not 7A6-negative cells, had apparent DNA fragments characteristic of cells undergoing apoptosis. By immunoblot, under reducing conditions, anti-7A6 detected a 38-kDa protein band in the cell lysates prepared from apoptotic cells. Immunoelectron microscopy showed the 7A6 Ag to be localized to the membrane of mitochondria in apoptotic Jurkat cells. These results indicate that anti-7A6 defines a novel epitope on the mitochondrial membrane protein that appears to be exposed on cells undergoing apoptosis, suggesting that the 7A6 molecule may be involved in the molecular cascade of apoptotic cell death.

Our previous studies demonstrated that upon activation, monocytes (Mo) were able to sensitize peripheral blood T (PBT) cells to apoptosis induced by treatment with PMA. However, it is unknown what gene products provide the death signal to the sensitized PBT cells and how activated Mo enable PBT cells to become susceptible to apoptosis. Here, we show that PBT cells, but not Mo, express functional Fas ligand upon treatment with PMA. Moreover, this Mo-dependent T cell apoptosis could be blocked by a Fas-Ig fusion protein, as well as by a nonlytic mAb against Fas molecule. These results strongly suggest involvement of Fas-Fas ligand interaction in the death of PBT cells. Unlike Fas-induced apoptosis, however, Mo-dependent T cell death was completely inhibited by overexpression of the Bcl-2 protein, and PMA alone was sufficient to trigger apoptosis in T cells when Mo were included in culture. Furthermore, anti-CD11a, anti-CD18, or anti-CD45/CD45RA mAbs; could prevent PBT cells from death triggered by PMA plus Mo, suggesting that these Ags participate in the apoptotic process. The participation of CD45RA in the death of PBT cells was further demonstrated by the observation that the J45.01 cell line, a CD45-deficient variant of Jurkat cells, did not undergo apoptosis by this Mo-dependent mechanism. When transfected with cDNA encoding CD45RA, J45.01 cells acquired apoptotic response to PMA stimulation in the presence of Mo to a similar, but lesser, degree than normal Jurkat cells.

This study demonstrates that the 175-kDa form of dipeptidyl peptidase IV (DPPIV) found in normal human serum is identical with a similarly-sized Ag, DPPT-L, found to be rapidly expressed on the surface of activated T cells. As activation progresses, the expression of DPPT-L reaches a peak on day 3, after which expression falls, whereas expression of the 105-kDa CD26/DPPIV detected by the mAb 1F7 increases, as does the ability to bind adenosine deaminase. The loss of DPPT-L from the surface of activated T cells correlates exactly with the appearance of DPPT-L and DPPIV activity in serum-free tissue culture medium. The release of DPPIV was generally greater from CD4+ cells than from CD8+ T cells, and within the CD4+ subset, the CD45RO+ subset was the major source, which correlated with surface expression before culture. We show that the DPPIV released from activated T cells is antigenically, biochemically, and enzymatically similar to DPPIV circulating in the serum and is distinct from the DPPIV activity of 105-kDa CD26. The T cell-released DPPIV is able to function as a costimulating molecule for the response to the recall Ag, tetanus toxoid, at levels similar to those at which recombinant soluble CD26 and serum DPPIV exhibit costimulatory function, suggesting that the released DPPIV may serve an important immunoregulatory function in vivo, both locally and within the systemic circulation.

CD26, a T cell activation Ag, also known as dipeptidyl peptidase IV, is directly associated with adenosine deaminase (ADA) on the surface of T cells and T cell lines. In the present study, we examined both the binding of ADA and CD26 and the functional consequences of this interaction. We found that ADA was associated with CD26 on T cell lines lacking either ADA or dipeptidyl peptidase IV enzymatic activity, indicating that the association between dipeptidyl peptidase IV and ADA did not require enzymatic activity. Moreover, using immunoelectron microscopy, we demonstrated that CD26 and ADA co-localized on the cell surface, but not inside cells, suggesting that CD26 did not transport ADA to the surface. In keeping with this observation, we showed that human CD26-transfected murine pre-B cell lines lacking human ADA acquired ADA from an extracellular source. More importantly, adenosine in the absence of cell surface ADA inhibited T cell proliferation and IL-2 production induced by various stimuli. On the other hand, cells expressing ADA and CD26 on the surface were much more resistant to the inhibitory effect of adenosine. These data suggest that ADA on the cell surface is involved in an important immunoregulatory mechanism by which released ADA binds to cell surface CD26, and this complex is capable of reducing the local concentration of adenosine.

Human CD26, a Type II membrane glycoprotein with intrinsic dipeptidylpeptidase IV (DPPIV) activity and ability to bind adenosine deaminase type I (ADA-1), is expressed on epithelial cells constitutively, but on T lymphocytes its expression is regulated. A soluble form of CD26/DPPIV has been described in plasma and related to immunological status, but it has been defined by the presence of DPPIV activity rather than by isolation. Using nondenaturing chromatographic techniques followed by nondenaturing native preparative electrophoresis, we obtained a homogeneous preparation of soluble serum DPPIV and compared it with a recombinant soluble CD26/DPPIV (rsCD26). We show that serum DPPIV is a monomer of 175 kDa in contrast to rsCD26 of 105-110 kDa, that it exists as a trimer, and that it is probably a serine proteinase. Deglycosylation removed N-linked sugar from both serum DPPIV and rsCD26; no O-linked glycosylation was observed, revealing a protein core of 130 kDa for serum DPPIV. The large serum form expresses functional DPPIV activity with substrate and inhibitor specificities and pH activity profile similar to those of rsCD26. Epitope analysis showed that monoclonal antibodies against five epitopes expressed by rsCD26 also bound, but more weakly, with serum DPPIV. Analysis of peptides after limiting proteolysis and N-terminal sequences reveals no homology with rsCD26 but some identity with other peptidases. Unlike rsCD26, the serum form does not bind ADA-1 and has no ADA-1 already associated with it. Similarly to rsCD26, serum DPPIV is a potent T cell costimulator. We conclude that the serum form of DPPIV is unique and is not a breakdown product of membrane CD26. The conservation of DPPIV activity and five epitopes specific to rsCD26 suggest, however, a significant structural similarity.

The engagement of CD27 with its ligand CD70 is considered to play an important role in T cell costimulation. In the present study, we investigated both the kinetics of CD70 expression and the contribution of its interaction with CD27 in T cell immune responses. CD70 was found to be expressed almost equally on both activated CD4 and CD8 T cells. On subsets of CD4 T cells, however, CD70 expression was induced preferentially on the CD45R0 T cell population after activation, whereas its expression was not noted on CD45RA T cells for almost 2 wk following activation. In long-term culture with media containing T cell growth factor (TCGF) and rIL-2, the expression of CD70 was increased markedly on CD45R0 T cells and minimally expressed on CD45RA T cells. In addition, strong surface expression of CD70 was observed on T cell clones originally derived from CD45R0+ CD4 T cells, whereas T cell clones originally derived from CD45RA+ CD4 T cells showed lower levels of expression. The addition of irradiated, activated CD45R0 T cells to CD45RA T cells caused a down-regulation of CD27 expression and an up-regulation of CD25 expression. These changes were blocked by addition of the anti-CD70 mAb, suggesting that direct contact between CD45R0 T cells and CD45RA T cells via CD27/CD70 occurred, leading to the activation of CD45RA T cells as measured by CD25 expression. These observations strongly support the notion that the engagement of CD27 plays an important regulatory role in the communication of subsets of CD45R0 and CD45RA T cells.

Patients who have undergone allogeneic bone marrow transplantation (allo-BMT) are susceptible to a variety of opportunistic infectious complications in the months to years after engraftment. Impaired in vitro T-cell functions have been documented in these patients, and these T-cell dysfunctions contribute to the prolonged immune deficiency after allo-BMT. In the present study, we examined the expression of CD26 as well as the reconstitution of CD26-mediated T-cell costimulation via the CD3 and CD2 pathways at various times in patients aged greater than 18 years after CD6-positive, T-cell depleted allo-BMT. We found that the percentage of CD26- and CD3-positive cells, as well as the levels of expression of both antigens, was lower than in normal controls during the first 4 months after CD6-depleted allo-BMT. Subsequently, the amount of lymphocytes expressing CD3 and CD26 and the quantitative surface expression of CD3 and CD26 were not significantly different in patients and normal controls. Functional studies showed that CD26-mediated T-cell proliferation via the CD3 pathway was considerably improved and almost reached normal levels by 1 year, whereas recovery of CD26-mediated T-cell proliferation via the CD2 pathway was delayed for at least 2 years after CD6-depleted allo-BMT. As CD26 involvement in the regulation of human thymocyte activation is restricted preferentially to the CD3 pathway--unlike its involvement with both CD3 and CD2 pathways of peripheral T cells--our results suggest that the different effects of CD26-mediated costimulation via the CD3 and CD2 pathways after CD6-depleted allo-BMT may be a reflection of peripheral T-cell immaturity in those individuals, similar to that seen in mature medullary thymocytes or cord T lymphocytes.

CD27 is a 120-kDa transmembrane homodimeric molecule expressed on the majority of T cells, B cells, and NK cells that belongs to the TNFR/nerve growth factor receptor family. The interaction between CD27 and its ligand, CD70, is thought to play an important role in T cell activation. In this paper we have examined the signal-transducing potential of CD27 in T cell costimulation. Anti-CD27 mAb, anti-1A4, induced substantial proliferation of peripheral blood T cells in the presence of a suboptimal dose of PMA, phytohemagglutinin, anti-CD2, or anti-CD3 together with a second Ab to cross-link the CD27 molecule. This T cell proliferation was also observed by using CD70 transfectant cells. CD27 cross-linking maximally induced proliferation of CD45RA+CD4 T cells but only slightly induced proliferation of CD45RO+CD4 T cells. CD27-mediated T cell proliferation did not seem to be dependent on the IL-2/IL-2R system because no detectable level of IL-2 was secreted, and only a partial inhibition was observed with anti-IL-2 and anti-IL-2R Abs. Furthermore, an increase in intracellular Ca2+ was observed in PMA-treated T cells when the CD27 molecule was cross-linked. More importantly, CD27 ligation induced protein tyrosine phosphorylation, especially 70 kDa of cellular substrate, including ZAP-70, in T cells. Herbimycin A, a protein tyrosine kinase inhibitor, and staurosporine, a protein kinase C inhibitor, blocked T cell proliferation induced by CD27 ligation, suggesting the possibility that the activation of protein tyrosine kinase and protein kinase C is required for CD27-mediated T cell costimulation. These results clearly demonstrate that the CD27/CD70 interaction induces costimulatory signals in T cells, especially CD45RA+ naive T cells, indicating that CD27 serves as a T cell signal-transducing molecule.

CD27 is a disulfide-linked 120-kDa transmembrane glycoprotein expressed on the majority of T cells, B cells, and NK cells; it has homology to a family of molecules that includes the receptors for nerve growth factor and TNF. Previous studies strongly suggest that the CD27 molecule plays a key role in the process of T cell activation. To further determine its role in T cell activation, a recombinant soluble molecule composing only the extracellular domain of CD27 was produced by transfection of Chinese hamster ovary cells. We have defined the binding properties of recombinant soluble CD27 (rsCD27) to CD27 ligand (CD27L) cDNA transfected NIH 3T3 cells and have determined its functional effects on in vitro T cell activation as well as on PWM-driven B cell IgG synthesis. rsCD27 bound specifically to CD27L and the binding was inhibited by one of our anti-CD27 mAbs, anti-1A4, suggesting that the 1A4 epitope of CD27 plays a role in the binding to CD27L. Functionally, rsCD27 inhibited T cell proliferation induced by various stimuli, such as PHA, tetanus toxoid, and anti-CD2, as well as PWM-driven B cell IgG synthesis, similar to the effects of adding anti-1A4. Determination of CD27L expression showed that CD27L mRNA is induced rapidly on activated T and B cells. Taken together, these results provide direct evidence that CD27-CD27L interaction plays a critical role in T cell activation as well as in T cell-dependent B cell IgG synthesis, suggesting that the CD27-CD27L interaction may constitute a component of the T cell-T cell or T cell-B cell interaction seen after activation with Ag or mitogen.

Antibodies to the CD6 Ag have been described as having pan-T cell reactivity. We have recently demonstrated, however, that after treatment of PBL with an anti-CD6-blocked ricin-conjugated immunotoxin, clonal populations of CD3+, CD6- cells can be identified. Herein we show that through dual parameter staining of freshly isolated E-rosette+ cells, an average of 5 to 6% of either CD3+ or CD5+ cells express little or no CD6 on their surface. After negative selection by antibody-coated paramagnetic bead depletion, expanded CD6- T cells were shown to be CD1a-, CD2+, CD3+, CD5+, CD16-, CD56-, TCR-gamma/delta-, and consisted of both CD4+ and CD8+ cells. Furthermore, staining of digitonin permeabilized cells showed no cytoplasmic expression of the CD6 Ag and CD6 mRNA was not detected by Northern blot analysis. Identical staining patterns were observed for T cell clones isolated through bead depletion or immunotoxin treatment and expanded with either PHA or immobilized anti-CD3 mAb. It was also found that, relative to unfractionated T cells, the surface expression of CD5 was significantly diminished on CD6- T cells. Functionally, freshly isolated CD6- T cells showed substantially reduced alloreactivity in MLR compared with unfractionated E-rosette+ cells, yet both gave similar proliferative responses to either PHA or soluble tetanus toxin Ag. We conclude that there exists a minor subpopulation of mature T cells in peripheral blood that lack CD6. The diminished alloreactivity of these cells may help to explain the low incidence of graft-vs-host disease, despite high levels of engraftment, that has been reported in allogeneic bone marrow transplant patients receiving marrow treated with anti-CD6 (T12) mAb plus C'.

We have developed a bispecific antibody that recognizes the CD4 and CD26 antigens simultaneously and that was examined for its ability to target CD4+CD26+T cells. These latter cells constitute the activated component of the CD4+ CD29highCD45RO+ memory T-cell subset that provides help for B-cell Ig synthesis and help for responses against recall antigens. The purified bispecific antibody exhibited an estimated dissociation constant (kd) of 2.4 x 10(-9) mol/L, on comparison with 1.1 x 10(-9) mol/L for anti-CD26, and 1.6 x 10(-10) mol/L for anti-CD4. Surface plasmon resonance was used to show the bifunctional capacity of the antibody. On binding 125I-bispecific antibody to phytohemagglutinin (PHA)-activated T cells, 54.4% of the bound antibody was internalized. This was the result of bispecific binding, because monovalent fragments of anti-CD4 and anti-CD26 were not able to modulate antigen or induce internalization using both a fluorescent assay and an 125I-internalization assay. The ability of the bispecific antibody to be internalized was used to deliver a toxin, blocked ricin, specifically to cells that are CD4+CD26+. The inability of monovalent fragments to be internalized formed the basis for our hypothesis that monovalent binding by the bispecific immunotoxin would not result in internalization. Against resting E+ T cells, the bispecific immunotoxin developed a minimal effect. On preactivating the same cells, using phorbol myristate acetate (PMA)/ionomycin on concanavalin A (ConA) or especially PHA, levels of CD26 were upregulated and the immunotoxin effectively inhibited the ability to provide help for B-cell Ig synthesis while leaving intact the CD4-CD26+ and CD4+CD26- populations; an effect observed both functionally and by phenotype. The bispecific antibody proved to be most effective at inhibiting a heterologous mixed leukocyte reaction. We propose that this reagent may form the basis for the rational design of toxins designed to modulate activated T cells from, or directed against, tissue grafts.

The human CD4 population can be divided into functionally distinct and largely reciprocal subsets based on their differential expression of CD45 isoforms (CD45RA, CD45RO) and the CD29/VLA beta chain. CD4+CD45RO+ CD29high "memory" (helper inducer) cells respond maximally to recall antigens and provide help for B cell IgG synthesis. In contrast, the CD4+CD45RA+ CD29low "naive" (suppressor inducer) population responds poorly to recall Ag, lacks helper function for B cells, but can both induce CD8 cells to suppress B cell IgG synthesis and proliferate preferentially in an autologous mixed lymphocyte response (AMLR). The phenotypic "conversion" after activation and the preferential responsiveness of CD45RA-CD45RO+ CD29high cells to recall antigen led to the view that CD45RA+ cells are "naive" and immature and convert to CD45RA-CD45RO+ "memory" cells after activation. This conversion was believed by many to be unidirectional and irreversible. It has become increasingly clear that the naive-memory concept outlined above is far from settled and that naive CD4+CD45RA+ T cells retain their unique functional program after activation and are distinct from the freshly isolated CD4+CD45RO+ subset. Moreover, CD45RA is not irreversibly lost following activation, but in fact recycles on the cell surface. Given the problems with CD45 isoform expression as a definition of maturational state, we have investigated the possibility that more reliable cell surface molecules are needed which could delineate between the functions of activated CD45RA+ and CD45RA- CD45RO+ cells. We could show that CD31 and CD27 are preferentially expressed on the CD4+CD45RA+ subset of cells and their expressions are stably maintained on these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The CD27 Ag is expressed by the majority of resting T lymphocytes and appears to play a crucial role in T cell activation. We found that some resting peripheral blood NK cells also express CD27. Furthermore, CD27 expression was up-regulated on NK cells stimulated by IL-2. The cytolytic activity of IL-2-activated, but not resting, NK cells was inhibited by an anti-CD27 mAb (anti-1A4). However, anti-1A4 did not affect conjugate formation between IL-2-activated NK cells and tumor cell targets. In contrast, anti-1A4 inhibited CD2-mediated calcium mobilization and the serine esterase activity of NK cell granules. These inhibitory effects could be mediated in part by increase in intracellular cAMP levels induced by anti-1A4. Our results suggest that the CD27 Ag plays an important role in the regulation of activated NK cells.

A cDNA encoding the T cell activation Ag CD26 was isolated from human PHA-activated T cells by using an expression cloning method. The nucleotide sequence obtained predicts a protein of 766 amino acids of type II membrane topology, with six amino acids in the cytoplasmic region. The predicted amino acid sequence of the Ag was 85% homologous to that of the dipeptidyl peptidase IV enzyme isolated from rat liver. Derivatives of the human leukemic T cell line Jurkat transfected with a CD26 expression plasmid were established. Characterization of the CD26 Ag expressed by the transfected Jurkat cells revealed that the Ag could be immunoprecipitated as a 110-kDa molecule similar to that found on peripheral blood T cells and that the Ag had dipeptidyl peptidase IV activity. Functional analysis of these Jurkat transfectants showed that cross-linking of the CD26 and CD3 Ag with their respective antibodies resulted in enhanced intracellular calcium mobilization and IL-2 production. These results provide direct evidence that the CD26 Ag plays a role in T cell 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.