Differential costimulatory effects of adhesion molecules B7, ICAM-1, LFA-3, and VCAM-1 on resting and antigen-primed CD4+ T lymphocytes.

Optimal proliferation of T cells although initiated via ligation of the CD3/TCR complex requires additional stimulation resulting from adhesive interactions between costimulatory receptors (R) on T cells and their counter-R on APC. At least four distinct adhesion molecules (counter-R) present on APC, B7, ICAM-1 (CD54), LFA-3 (CD58), and VCAM-1 have been individually shown to costimulate T cell activation. Because some of these molecules may be expressed simultaneously on APC, it has been difficult to examine relative contributions of individual counter-R during the induction of T cell proliferation. We have produced soluble IgC gamma 1 fusion chimeras (receptor globulins or Rg) of B7, ICAM-1, LFA-3, and VCAM-1 and compared their relative abilities to costimulate proliferation of resting or Ag-primed CD4+ T cells. When co-immobilized with mAb directed at TCR alpha beta or CD3 but not CD2 or CD28, each Rg induced proliferation of both resting and Ag-primed CD4+ cells. In contrast, similarly co-immobilized CD7 Rg or ELAM-1 Rg were ineffective. Resting CD4+ T cells produced more IL-2, expressed significantly higher levels of IL-2R alpha, and proliferated more efficiently when costimulated with either ICAM-1 Rg or VCAM-1 Rg than with B7 Rg or LFA-3 Rg. CD4+ CD45RO+ memory T cells proliferated more vigorously in response to the costimulation by each of the four Rg than CD4+ CD45RA+ naive T cells. In contrast with the behavior of resting CD4+ T cells, proliferation of Ag-preactivated CD4+ T cells was most efficient when costimulated by B7 Rg. The costimulatory effect of LFA-3 Rg on Ag-primed CD4+ T cells was weaker than that of B7 Rg but was significantly greater than that of either ICAM-1 Rg or VCAM-1 Rg. These results suggest that resting and Ag-primed CD4+ T cells preferentially respond by proliferation to different costimulatory counter-R. ICAM-1 and VCAM-1 may be involved in the initiation of proliferation of Ag-responsive T cells, and B7 and LFA-3 may facilitate sustained proliferation of Ag-primed T cells. The cumulative costimulation by the above counter-R may facilitate optimal expression of various regulatory and effector functions of T cells.     

Interaction of CD2 with its ligand, LFA-3, in human T cell proliferation

Recently, it has been demonstrated that lymphocyte function-associated Ag (LFA-3) is a natural ligand for CD2 and that this receptor-ligand interaction functions in cell-cell adhesion. In this report, we demonstrate that LFA-3 plays a role in T cell activation. L cells were transfected with human genomic DNA and sorted for expression of LFA-3. We demonstrate that LFA-3+ L cells, together with anti-CD3 mAb or with suboptimal doses of PHA, stimulate proliferation of human peripheral blood T cells. Furthermore, thymocyte proliferation was induced by LFA-3+ L cells and suboptimal doses of PHA. Proliferation was inhibited by mAb directed against either CD2 or LFA-3. Stimulation of thymocytes by the combination of PHA and LFA-3+ L cells resulted in the increased expression of the IL-2R, as well as of the surface Ag 4F2, transferrin receptor, and HLA-DR. These data support the conclusion that LFA-3 plays a role in CD2-dependent T cell activation. LFA-3 is widely distributed and is expressed on all APC and target cells. Thus, the ability of the CD2/LFA-3 interaction to costimulate with an anti-CD3 mAb suggests that the CD2/LFA-3 interaction may be involved not only in an Ag-independent alternate pathway of T cell activation but also in Ag-specific T cell activation.     

Regulation of adhesion molecule expression by CD8 T cells in vivo. I. Differential regulation of gp90MEL-14 (LECAM-1), Pgp-1, LFA-1, and VLA-4 alpha during the differentiation of cytotoxic T lymphocytes induced by allografts.

A variety of adhesion molecules regulate the traffic and tissue localization of lymphocytes in vivo by mediating their binding to vascular endothelial cells. The homing receptor gp90MEL-14 (gp90), also known as LECAM-1 or L-selectin, mediates the adhesion of lymphocytes to specialized high endothelial venules in lymph nodes (LN) and is the primary molecule regulating lymphocyte recirculation and homing to LN, whereas other adhesion molecules have a major role in the localization of lymphocytes in inflammatory sites. We used four-color flow cytometric analysis to examine the regulation of adhesion receptor expression on LN CD8 T cells responding to skin allografts in vivo. In normal mice, greater than 95% of LN CD8 T cells are gp90+, being either gp90+Pgp1- (Population (Pop.) 1 or gp90+Pgp-1+ (Pop.2). Allografting induces the down-regulation of gp90 and up-regulation of Pgp-1 on a subset of cells, resulting in the appearance of CD8+gp90-Pgp-1hi (Pop. 3) cells. Pop. 3 cells also express high levels of LFA-1, ICAM-1, and ICAM-2, and a subset of them are VLA-4 alpha-positive. Purified Pop. 3 cells have potent cytolytic activity directed against donor alloantigen, whereas no such activity is present in Pop. 1 or 2 cells. Correlating with this is the high granzyme activity in Pop. 3 cells. In addition, Pop. 3 lymphocytes, but not Pop. 1 or 2, secrete a large amount of IFN-gamma in response to Ag. Finally, the CD8 T cells that infiltrate sponge matrix allografts are markedly enriched for the Pop. 3 subset. These results show that, during the immune response to alloantigen in vivo, a small subset of CD8 T cells down-regulates the LN homing receptor while increasing the expression of other adhesion molecules, as they differentiate into highly active cytolytic T lymphocytes. Thus, the differential regulation of LN homing receptors and receptors for peripheral vascular endothelium provides a mechanism that would redirect the traffic of activated effector cells away from lymphoid tissue and to sites of Ag deposition, where they would participate in the inflammatory response.     

Stimulation of IFN-gamma, TNF-alpha, and TNF-beta secretion in IL-2-activated T cells: costimulatory roles for LFA-1, LFA-2, CD44, and CD45 molecules.

Lymphokine-activated killer (LAK) cells are peripheral blood lymphocytes (PBLs) that possess the ability to kill target cells in a non-major histocompatibility complex (MHC)-restricted manner. Both NK and T cells can be stimulated with interleukin-2 (IL-2) to become LAK cells. We previously reported that the interaction of LAK cells with tumor cells also induces the secretion of interferon-gamma (IFN-gamma). The NK subset of LAK (LAK-NK) cells is stimulated by tumor cells to secrete IFN-gamma in a non-MHC-restricted manner while the T cell subset of LAK (LAK-T) cells is stimulated to secrete IFN-gamma upon cross-linking of the T cell receptor (TCR)-CD3 complex. We here report that LAK-T cells stimulated with anti-CD3 mAbs and tumor cells secrete two additional cytokines, tumor necrosis factor-alpha (TNF-alpha) and TNF-beta/lymphotoxin (TNF-beta). In addition, we demonstrate that at least four other structurally unrelated molecules, in addition to the TCR-CD3 complex, on LAK-T cells participate in the stimulation of IFN-gamma, TNF-alpha, and TNF-beta production. These molecules are the lymphocyte function associated antigen-1 (LFA-1), lymphocyte function associated antigen-2 (LFA-2), CD44, and CD45. LFA-1 is an integrin, LFA-2 is a member of the immunoglobulin supergene family, CD44 is homologous to the cartilage link proteins, and CD45 is a tyrosine phosphatase. Ligands to three of these molecules have been identified; ICAM-1, LFA-3, and hyaluronic acid binding to LFA-1, LFA-2, and CD44, respectively. LFA-1, LFA-2, and CD44 are reported to function both as adhesion molecules and as costimulators in resting T cells. Our data suggest that these three molecules enhance IFN-gamma, TNF-alpha, and TNF-beta production by augmenting LAK-T cell to tumor cell adhesion and also by functioning as costimulators.     

Loss of CD45R (Lp220) represents a post-thymic T cell differentiation event

CD45R+ and CDw29+ CD4+ T cells are widely regarded as separate functionally defined T cell lineages. The work described here indicates that they represent maturation stages within the same differentiation pathway. Purified populations of CD4+ or CD8+ T cells, after stimulation with PHA, lose cell surface expression of CD45R (Lp220) and gain an increased surface density of CDw29 (4B4). Clonal analysis demonstrated that individual CD4+ CD45R+ T cells lost CD45R and acquired CDw29 with time in culture. This effect was selective for the high Mr 220-kDa form of the T200 (CD45) complex because the density of CD45, detected by an antibody to common determinants, did not decrease. This strongly indicates that CD45R+ cells are an immature stage in a lineage that culminates in CDw29 expression. To further define the expression of CD45R and CDw29, we analyzed infant thymus cells. Thymocytes include only 4 to 6% CD45R+ cells, but 95% express CDw29 in moderate density. The CD45R+ set appears to include mainly single CD4+ or CD8+, CD3 "bright" medullary cells, although only 15 to 25% of thymocytes with medullary phenotype express CD45R. In vitro culture of thymocytes with Con A and T cell growth factor induces expression of CD45R but these cells differ from the peripheral CD45R+ set by virtue of their co-expression of a high density of CDw29 (4B4) Ag. We postulate that post-thymically CD45R (Lp200) and CDw29 (4B4) comprise a functional assembly on the surface of T cells that changes in composition after stimulation with Ag or mitogen. This may result in enhanced ability of an Ag-experienced T cell to respond effectively to Ag due perhaps to a more efficient signaling complex.     

Role of LFA-1 and VLA-4 in the adhesion of cloned normal and LFA-1 (CD11/CD18)-deficient T cells to cultured endothelial cells. Indication for a new adhesion pathway.

Patients with the leukocyte adhesion deficiency (LAD) syndrome have a genetic defect in the common beta 2-chain (CD18) of the leukocyte integrins. This defect can result in the absence of cell surface expression of all three members of the leukocyte integrins. We investigated the capacity of T cell clones obtained from the blood of an LAD patient and of normal T cell clones to adhere to human umbilical vein endothelial cells (EC). Adhesion of the number of LAD T cells to unstimulated EC was approximately half of that of leukocyte function-associated antigen (LFA)-1+ T cells. Stimulation of EC with human rTNF-alpha resulted in an average 2- and 2.5-fold increase in adhesion of LFA-1+ and LFA-1- cells, respectively. This effect was maximal after 24 h and lasted for 48 to 72 h. The involvement of surface structures known to participate in cell adhesion (integrins, CD44) was tested by blocking studies with mAb directed against these structures. Adhesion of LFA-1+ T cells to unstimulated EC was inhibited (average inhibition of 58%) with mAb to CD11a or CD18. Considerably less inhibition of adhesion occurred with mAb to CD11a or CD18 (average inhibition, 20%) when LFA-1+ T cells were incubated with rTNF-alpha-stimulated EC. The adhesion of LFA-1- T cells to EC stimulated with rTNF-alpha, but not to unstimulated EC, was inhibited (average inhibition, 56%) by incubation with a mAb directed to very late antigen (VLA)-4 (CDw49d). In contrast to LAD T cell clones and the LFA-1+ T cell line Jurkat, mAb to VLA-4 did not inhibit adhesion of normal LFA-1+ T cell clones to EC, whether or not the EC had been stimulated with rTNF-alpha. We conclude that the adhesion molecule pair LFA-1/intercellular adhesion molecule (ICAM)-1 plays a major role in the adhesion of LFA-1+ T cell clones derived from normal individuals to unstimulated EC. Adhesion of LFA-1-T cells to TNF-alpha-stimulated EC is mediated by VLA-4/vascular cell adhesion molecule (VCAM)-1 interactions. Since we were unable to reduce significantly the adhesion of cultured normal LFA-1+ T cells to 24 h with TNF-alpha-stimulated endothelium with antibodies that block LFA-1/ICAM-1 or VLA-4/VCAM-1 interactions, and lectin adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 appeared not to be implicated, other as yet undefined cell surface structures are likely to participate in T cell/EC interactions.     

Phenotypic identification of memory cytolytic T lymphocytes in a subset of Lyt-2+ cells

Murine peripheral Lyt-2+ T cells could be subdivided according to surface expression of the Pgp-1 glycoprotein into major (71%) Pgp-1- and minor (29%) Pgp-1+ subsets. A striking correlation was observed between Pgp-1 expression and enrichment for antigen-specific memory cytolytic T lymphocyte precursors (CTLp). After immunization with the male minor transplantation antigen H-Y, virtually all the H-Y-specific CTLp were found in the minor Pgp-1+ subset of Lyt-2+ cells. In addition, after alloimmunization the frequency of allospecific CTLp resistant to inhibition by anti-Lyt-2 antibody was markedly enriched within the Pgp-1+ cells, suggesting an enrichment for CTLp bearing high avidity antigen receptors. Taken together, these data suggest that surface Pgp-1 expression is stably acquired at the time of primary antigenic stimulation by virgin T cells. As such, Pgp-1 represents an important marker for identifying a subset of Lyt-2+ T cells with the quantitative and qualitative properties of memory CTLp.     

The role of LFA-1/ICAM-1 interactions during murine T lymphocyte development.

We have examined the expression and function of the cell adhesion molecules LFA-1 (CD11a/CD18), ICAM-1 (CD54), and ICAM-2 in murine fetal thymic ontogeny and in the adult thymus. On fetal days 14 and 15, 40 to 50% of thymocytes coexpress high levels of LFA-1 and ICAM-1, as determined by flow cytometry. By day 16, more than 90% of fetal thymocytes are LFA-1+ ICAM-1hi, and all IL-2R+ cells are located in this population. Although LFA-1 expression remains unchanged thereafter, ICAM-1 expression appears to be differentially regulated in different thymocyte subpopulations, with CD4+8+ cells being ICAM-1lo and CD4-8- thymocytes remaining ICAM-1hi. ICAM-2 surface expression is dull on both fetal and adult thymocytes. Surprisingly, the expression of ICAM-1 is differentially up-regulated on T cells having a mature phenotype in thymus and in peripheral lymphoid organs, with CD8+ T cells bearing the highest amount of surface ICAM-1. Addition of anti-ICAM-1 or anti-LFA-1 antibodies to fetal thymic organ cultures results in the impaired generation of CD4+8+ cells. These results indicate that LFA-1/ICAM-1 interactions facilitate murine thymic development and suggest that cell adhesion molecules mediate important events in T cell differentiation.     

Somatostatin down-regulates LFA-1 activation by modulating Rap1 expression in CD4+ and CD8+ T cells

Leukocyte function-associated antigen-1 (LFA-1) is one of the integrins that are expressed on the leukocytes, and has been shown to play an important role in leukocyte trafficking. The adhesive activity of LFA-1 is governed partially by the Rap1. This study examined that the relationship between LFA-1 and Rap1 mRNA expressions by anti-CD3 and anti-CD3+SOM treatment in the CD4+ and CD8+ T cells. The LFA-1 mRNA expression levels following the anti-CD3 and anti-CD3+SOM treatment for 30 min was greater on the CD8+ T cells, and the LFA-1 expression of the CD8+ T cells with anti-CD+SOM treatment was affected more severely than that of the CD4+ T cells. The Rap1 mRNA expression patterns following anti-CD3 and anti-CD3+SOM stimulation in the CD4+ and CD8+ T cells were similar to the LFA-1 expression patterns, and the expression level following anti-CD3+SOM treatment was suppressed more significantly in the CD8+ T cells. These results suggest that the difference in the Rap1 expression level after stimulation might explain the differences in the LFA-1 expression level on the T cell subsets, and that the down-regulation of Rap1 expression following SOM treatment is closely related to the diminished LFA-1 expression.     

Age-related changes in LFA-1 expression, cell adhesion, and PHA-induced proliferation by lymphocytes from senescence-accelerated mouse (SAM)-P/8 and SAM-R/1 substrains.

Accelerated senescence-prone mice of the SAM-P/8jf series were compared with senescence-resistant SAM-R/1 controls in terms of age-related changes in phytohemagglutinin (PHA) proliferative responses and lymphocyte function-associated antigen-1 (LFA-1) utilization by non-adherent splenocytes. Advancing age was associated with a reduction in cell proliferative responses to PHA in both substrains, although the rate of decline was significantly more rapid in the senescence-prone animals. Conversely, in both substrains there was a progressive age-related increase in the proportion of splenocytes expressing high levels of LFA-1, and a parallel increase in the degree of LFA-1-dependent cell aggregation induced by phorbol ester. Age-matched SAM-P/8jf and SAM-R/1 mice did not differ in terms of LFA-1 expression or LFA-1-dependent cell aggregation. Two-color cytofluorometric analysis demonstrated the enhanced expression of LFA-1 expression by cells bearing the Pgp-1hi phenotype characteristic of memory lymphocytes. These results suggest that age-associated changes in lymphocyte adhesion are attributable to alterations in the relative numbers of memory cells expressing high levels of LFA-1, but are unlikely to contribute to the reduced proliferative response to mitogen in aged mice.     

The functional significance, distribution, and structure of LFA-1, LFA-2, and LFA-3: cell surface antigens associated with CTL-target interactions

Three cell surface antigens associated with the cytolytic T lymphocyte(CTL)-target cell interaction were identified by generation of monoclonal antibodies (MAb) against OKT4+, HLA-DR-specific CTL and selection for inhibition of cytolysis in a 51Cr-release assay. These MAb block cytolysis by both OKT4+ and OKT8+ CTL and the proliferative responses to PHA and the mixed lymphocyte response (MLR). LFA-1 is an antigen widely distributed on lymphoid tissues and is composed of two polypeptides of 177,000 and 95,000 Mr on all cell types studied. Anti-LFA-1 MAb block NK cell-mediated cytolysis in addition to T lymphocyte-mediated cytotoxicity and proliferation. LFA-2 (Mr = 55,000 to 47,000), a determinant on the sheep red blood cell receptor, is expressed by T cells but not B cells and appears specific for T cell functions. LFA-3 (Mr = 60,000) is a widely distributed antigen present on both hematopoietic and nonhematopoietic tissues and appears to only be involved in T cell functions. MAb to LFA-1 and LFA-2 inhibit function by binding to effector cell surface molecules, whereas anti-LFA-3 MAb appear to block by binding to the target cells. Together with previously described molecules, LFA-1, LFA-2, and LFA-3 demonstrate the complexity of CTL-mediated cytotoxicity at the molecular level.     

Loss of CD45R and gain of UCHL1 reactivity is a feature of primed T cells

A group of mAb recognizing the 200- and/or 220-kDa determinants (CD45R) of the leukocyte common Ag such as 2H4, WR16, MD4.3, and SN130 cross-block each other showing that they recognize a closely related epitope. The antibody UCHL1 reacts with a 180-kDa determinant of the leukocyte common Ag and exhibits a reciprocal T subset distribution pattern to the CD45R group. Peripheral blood T cells were 40% positive for UCHL1 and 58 to 65% positive for the CD45R antibodies; less than 1% of cells stained for both. On activation of CD45R+,UCHL1- T cells by PHA, up to 40% of cells became positive for both CD45R and UCHL1 by day 3. By day 7, CD45R+,UCHL1- cells fell from 90 to less than 21% whereas UCHL1+,CD45R- cells rose from 2 to 93%. Conversely, PHA-stimulated UCHL1+,CD45R- cells remained UCHL1+,CD45R- during the 7 days in culture showing that phenotypic change was unidirectional from CD45R+ to UCHL1+. In primary allogeneic mixed lymphocyte reactions, activated CD45R+ T cells also showed a change to UCHL1+. When these cells were rechallenged by the original alloantigen, the UCHL1+ cells showed 7- to 20-fold greater proliferation than the CD45R+ cells on day 3 after rechallenge. The recovery of virtually all alloantigen induced secondary proliferative response in the UCHL1+,CD45R- T cell population suggests that UCHL1 identifies a primed population of T cells which may include memory cells.     

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

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

Simultaneous increased expression of E-rosette receptor (CD2, T11) and T cell growth factor receptor on human T lymphocytes during activation

The E-rosette receptor (CD2, T11) is a differentiation antigen expressed on immature and mature human T lymphocytes. Activation of T cells from human peripheral blood with phytohemagglutinin (PHA) or with monoclonal antibody to the CD3-Ti complex (anti-Leu-4) caused the expression of CD2 to increase 10- to 20-fold. Dual parameter correlated analyses with antibody to the T cell growth factor (TCGF) receptor (anti-Tac) and anti-CD2 antibody demonstrated that the increase in CD2 expression occurred at the same time and on the same cells that expressed the TCGF receptor after stimulation with PHA. The increased expression of CD2 and the initial expression of Tac were totally inhibited by cycloheximide, but were not affected by sufficient actinomycin-D to block the T cell proliferative response. The expression of CD2 was compared with the expression of CD4 and CD8, i.e., T cell differentiation antigens on cytotoxic/suppressor or helper T cells, respectively. Although virtually all of the small percentage of freshly isolated Tac+ peripheral blood cells belonged to the CD4+, CD8- subset, both CD4+ and CD8+ T cells were equivalently activated by PHA to express Tac. By 20-30 hr after activation, the expression of CD4 or CD8 was initially decreased 10-50%. Subsequently, the expression of CD4 and CD8 returned to the levels on resting T cells but did not increase further. Therefore, the increase in CD2 expression does not reflect a universal property of cell surface antigens on activated T lymphocytes.     

Increased proportion of CD4+CDw29+CD45R-UCHL-1+ lymphocytes in the cerebrospinal fluid of both multiple sclerosis patients and healthy individuals

In this study we confirm earlier reports of an increase of the proportion of T and CD4+ lymphocytes and a decrease of B and CD8+ lymphocytes in cerebrospinal fluid (CSF) as compared to peripheral blood (PB) in MS patients. In addition we now demonstrate that this difference between CSF and PB lymphocyte populations is of the same magnitude in healthy individuals suggesting that it is physiological and not associated with disease. Functionally distinct subsets of the T human helper cell (CD4+) population have previously been defined by the monoclonal antibodies 4B4 (CDw29), Leu-18 (CD45R), and UCHL-1. In the present investigation we demonstrate a selective increase in the proportion of CD4+CDw29+CD45R-UCHL-1+ lymphocytes in CSF as compared to PB of both MS patients and healthy individuals, which strongly indicates that also this enrichment is physiological rather than associated with disease. A possible relationship between this subset of CD4+ lymphocytes and T memory cells is discussed.     

Distinction of virgin and memory T lymphocytes. Stable acquisition of the Pgp-1 glycoprotein concomitant with antigenic stimulation

The Pgp-1 glycoprotein was identified on a minor (27%) subset of peripheral Lyt-2+ or L3T4+ T cells. In contrast, mature medullary-type thymocytes (Lyt-2+ L3T4-, Lyt-2- L3T4+) were nearly devoid of cells expressing detectable surface Pgp-1. The appearance of peripheral Pgp-1- T cells was found to be thymus dependent, as demonstrated by the diminished proportion of Pgp-1- T cells after thymectomy and their virtual absence in athymic nude mice. The subsequent acquisition of surface Pgp-1 was found to be a stable differentiation event occurring concomitantly with primary antigenic stimulation; selected Pgp-1- mature T cells from thymus or periphery acquired constitutive expression of Pgp-1 after stimulation in vitro with alloantigen or mitogens. These observations were extended by studies in vivo showing that immunization with various antigens augmented the percentage of Pgp-1+ spleen cells within the Lyt-2+ subset. Furthermore, the frequencies of antigen-specific CTLp, after immunization by any of three different antigens tested, were greatly enriched in the Pgp-1+ compared with the Pgp-1- subpopulations. Peritoneal exudate Lyt-2+ cells, after a localized allograft rejection, demonstrated a particularly prominent Pgp-1+ subpopulation (78%) that contained virtually all the allospecific cytolytic activity. A model consistent with all of these data proposes that mature thymocytes lacking surface Pgp-1 upon emigration to the periphery acquire its expression at the time of primary antigenic stimulation. Hence, expression of Pgp-1 among peripheral T cells is an important differentiation marker for identifying antigen-stimulated memory T cells.     

LFA-3 plasmid DNA enhances Ag-specific humoral- and cellular-mediated protective immunity against herpes simplex virus-2 in vivo: involvement of CD4+ T cells in protection

Adhesion molecules are important for cell trafficking and delivery of secondary signals for stimulation of T cells and antigen-presenting cells (APCs) in a variety of immune and inflammatory responses. Adhesion molecules lymphocyte function-associated antigen (LFA)-1 and CD2 on T cells recognize intercellular adhesion molecule (ICAM)-1 and LFA-3 on APCs, respectively. Recent studies have suggested that these molecules might play a regulatory role in antigen-specific immune responses. To investigate specific roles of adhesion molecules in immune induction we coimmunized LFA-3 and ICAM-1 cDNAs with a gD plasmid vaccine and then analyzed immune modulatory effects and protection against lethal herpes simplex virus (HSV)-2 challenge. We observed that gD-specific IgG production was enhanced by LFA-3 coinjection. However, little change in IgG production was observed by ICAM-1 coinjection. Furthermore, both Th1 and Th2 IgG isotype production was driven by LFA-3. LFA-3 also enhanced Th cell proliferative responses and production of interleukin (IL)-2, interferon-gamma, IL-4, and IL-10 from splenocytes. In contrast, ICAM-1 showed slightly increasing effects on T-cell proliferation responses and cytokine production. beta-Chemokine production (RANTES, MIP-1alpha, and MCP-1) was also influenced by LFA-3 or ICAM-1. When animals were challenged with a lethal dose of HSV-2, LFA-3-coimmunized animals exhibited an enhanced survival rate, as compared to animals given ICAM-1 or gD DNA vaccine alone. This enhanced protection appears to be mediated by CD4+ T cells, as determined by in vitro and in vivo T-cell subset deletion. These studies demonstrate that adhesion molecule LFA-3 can play an important role in generating protective antigen-specific immunity in the HSV model system through increased induction of CD4+ Th1 T-cell subset.     

Triggering of co-mitogenic signals in T cell proliferation by anti-LFA-1 (CD18, CD11a), LFA-3, and CD7 monoclonal antibodies

Proliferative T cell responses were elicited in a comitogenic assay when purified mAb against CD 18, CD11a, LFA-3, and CD7 were immobilized onto solid plastic surfaces together with submitogenic doses of mAb against the CD3 complex. The proliferative response was associated to the production of IL-2 and to the expression of IL-2R. We explored the possibility that a second signal provided by either PMA or a Ca2+ ionofore could replace the anti-CD3 mAb in the comitogenic assay. Interestingly, our data clearly indicate that PMA but not the ionofore was capable of mediating the co-mitogenic effect in conjunction with solid-bound mAb (CDw18, CD11a, LFA-3, and CD7). We also demonstrate that the mAb (anti-CD4 and anti-CD2) which have been previously described as co-mitogenic in combination with anti-CD3 are capable of eliciting this activating signal in the presence of PMA. These data indicate that mAb to certain cell surface differentiation Ag that in soluble form inhibit T cell function such as LFA-1, LFA-3, and CD2 can under appropriate conditions induce co-mitogenic signals on T cells. Our results support the hypothesis that several cell surface differentiation Ag may participate in conjunction with the T3-Ti complex in the transmembrane signal transduction leading to T cell activation.     

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

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