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.     

Antigen-presenting cells containing multiple costimulatory molecules promote activation and expansion of human antigen-specific memory CD8<Superscript>+</Superscript> T cells

We have previously demonstrated that multiple immunizations with vector-based vaccines containing transgenes for tumor Ags and a triad of costimulatory molecules (TRICOM) enhance the expansion and functional avidity of Ag-specific memory CD8⁺ T cells in a mouse model. However, the effect of enhanced costimulation on human memory CD8⁺ T cells is still unclear. The study reported here was an in vitro investigation of the proliferation and function of CEA-specific human memory CD8⁺ T cells following enhanced costimulation. Our results demonstrated that TRICOM costimulation enhanced production of multiple cytokines and expansion of CEA-specific memory CD8⁺ T cells. The lytic capacity of memory CTLs toward CEA⁺ tumors was also significantly enhanced. IL-2Rα (CD25) was upregulated dramatically following APC-TRICOM stimulation, suggesting that the enhanced expansion of memory CD8⁺ T cells may be mediated by increased expression of IL-2R on memory T cells. The enhanced cytokine production and proliferation following TRICOM signaling was completely blocked by the combination of neutralizing Abs against B7-1, ICAM-1, and LFA-3, the costimulatory molecules comprising TRICOM. No difference in T-cell apoptosis was observed between APC-TRICOM and APC-wild-type groups, as determined by annexin V, Bcl-2, and active caspase-3 staining. Results indicated that enhanced costimulation greatly expanded human CEA-specific CD8⁺ T cells and enhanced T-cell function, without inducing increased apoptosis of CEA-specific memory CD8⁺ 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.     

A recombinant vector expressing transgenes for four T-cell costimulatory molecules (OX40L,B7-1, ICAM-1, LFA-3) induces sustained CD4+ and CD8+ T-cell activation,protection from apoptosis,and enhanced cytokine production

The role of OX40L on the activation of T cells was investigated using poxvirus vectors expressing OX40L alone or in combination with three other T-cell costimulatory molecules: B7-1, ICAM-1, and LFA-3. Poxvirus vector-infected cells were used to stimulate nai;ve or activated CD4(+) and CD8(+) T cells. These studies demonstrate that (a) OX40L plays a role in sustaining the long-term proliferation of CD8(+) T cells in addition to the known effect on CD4(+) T cells following activation, (b) OX40L enhances the production of Th1 cytokines (IL-2, IFN-gamma, and TNF-alpha) from both CD4(+) and CD8(+) while no change in IL-4 expression was observed, and (c) the anti-apoptotic effect of OX40L on T cells is likely the result of sustained expression of anti-apoptotic genes while genes involved in apoptosis are inhibited. In addition, these are the first studies to demonstrate that the combined use of a vector driving the expression of OX40L with three other costimulatory molecules (B7-1, ICAM-1, and LFA-3) both enhances initial activation and then further potentiates sustained activation of nai;ve and effector T cells.     

The LFA-1 ligand ICAM-1 provides an important costimulatory signal for T cell receptor-mediated activation of resting T cells

Functional studies demonstrate that T cell activation often requires not only occupancy of the TCR but costimulatory interactions of other molecules, which remain largely undefined. We have tested the hypothesis that LFA-1 interaction with its ligand intercellular adhesion molecule 1 (CD54) (ICAM-1) is such a costimulatory interaction in a model system using biochemically purified ICAM-1 and TCR cross-linking by anti-CD3 mAb OKT3 immobilized on plastic. Resting T cells do not respond to OKT3 mAb immobilized on plastic. However ICAM-1 deposited on plastic together with the nonmitogenic immobilized OKT3 results in a potent activating stimulus. This costimulation cannot be readily accounted for by ICAM-1-mediated adhesion but is consistent with a role in signaling, which is observed in ICAM-1-mediated augmentation of activation induced by PMA/ionomycin. The ability of ICAM-1 to costimulate with immobilized CD3 contrasts with minimal costimulatory activity of cytokines IL-1 beta, IL-2, and IL-6. The proliferative response to co-immobilized OKT3 and ICAM-1 is dependent on the IL-2R, which is induced only in the presence of both OKT3 and ICAM-1. The present data demonstrate that LFA-1/ICAM-1 interaction is a potent costimulus for TCR-mediated activation; this observation, interpreted in light of previous reports, suggests that LFA-1/ICAM-1 is of major physiologic importance as a costimulatory signal.     

Expanded CD8+ T cells of murine and human CLL are driven into a senescent KLRG1+ effector memory phenotype

Altered numbers and functions of T cells have previously been demonstrated in chronic lymphocytic leukemia (CLL) patients. However, dynamics and specific T-cell subset alterations have not been studied in great detail. Therefore, we studied CLL blood lymphocyte subsets of individual patients in a longitudinal manner. Dynamic expansions of blood CD4 ⁺ and CD8 ⁺ T-cell numbers were consistently associated with a progressively increasing CLL leukemic compartment. Interestingly, the T-cell subset expansion over time was more pronounced in CD38 ⁺ CLL. Additionally, we performed gene expression profiling of CD3 ⁺ T cells of CLL patients and normal donors. Using gene set enrichment analysis, we found significant enrichment of genes with higher expression in CLL T cells within CD8⁺ effector memory and terminal effector T-cell gene signatures. In agreement with these data, we observed a marked expansion of phenotypic CD8 ⁺ effector memory T cells in CLL by flow cytometry. Moreover, we observed that increments of CD8 ⁺ effector memory T cells in human CLL and also mouse CLL (Eμ-TCL1 model) were due to an expansion of the inhibitory killer cell lectin-like receptor G1 (KLRG1) expressing cellular subset. Furthermore, higher plasma levels of the natural KLRG1 ligand E-cadherin were detected in CLL patients compared to normal donor controls. The predominance of KLRG1⁺ expression within CD8⁺ T cells in conjunction with increased systemic soluble E-cadherin might significantly contribute to CLL immune dysfunction and might additionally represent an important component of the CLL microenvironment.     

Intra-articular CD1c-expressing myeloid dendritic cells from rheumatoid arthritis patients express a unique set of T cell-attracting chemokines and spontaneously induce Th1, Th17 and Th2 cell activity

Introduction

Myeloid dendritic cells (mDCs) are potent T cell-activating antigen-presenting cells that have been suggested to play a crucial role in the regulation of immune responses in many disease states, including rheumatoid arthritis (RA). Despite this, studies that have reported on the capacity of naturally occurring circulating mDCs to regulate T cell activation in RA are still lacking. This study aimed to evaluate the phenotypic and functional properties of naturally occurring CD1c (BDCA-1)⁺ mDCs from synovial fluid (SF) compared to those from peripheral blood (PB) of RA patients.

Methods

CD1c⁺ mDC numbers and expression of costimulatory molecules were assessed by fluorescence-activated cell sorting (FACS) analysis in SF and PB from RA patients. Ex vivo secretion of 45 inflammatory mediators by mDCs from SF and PB of RA patients was determined by multiplex immunoassay. The capacity of mDCs from SF to activate autologous CD4⁺ T cells was measured.

Results

CD1c⁺ mDC numbers were significantly increased in SF versus PB of RA patients (mean 4.7% vs. 0.6%). mDCs from SF showed increased expression of antigen-presenting (human leukocyte antigen (HLA) class II, CD1c) and costimulatory molecules (CD80, CD86 and CD40). Numerous cytokines were equally abundantly produced by mDCs from both PB and SF (including IL-12, IL-23, IL-13, IL-21). SF mDCs secreted higher levels of interferon γ-inducible protein-10 (IP-10), monokine induced by interferon γ (MIG) and, thymus and activation-regulated chemokine (TARC), but lower macrophage-derived chemokine (MDC) levels compared to mDCs from PB. mDCs from SF displayed a strongly increased capacity to induce proliferation of CD4⁺ T cells associated with a strongly augmented IFNγ, IL-17, and IL-4 production.

Conclusions

This study suggests that increased numbers of CD1c⁺ mDCs in SF are involved in the inflammatory cascade intra-articularly by the secretion of specific T cell-attracting chemokines and the activation of self-reactive T cells.     

Lack of ICAM-1 on APCs during T cell priming leads to poor generation of central memory cells

ICAM-1/LFA-1 interactions are known to enhance T cell/APC interactions and to promote T cell activation and cytokine secretion. We have analyzed the consequences of ICAM-1-mediated signaling on the generation of memory T cell subsets. We report that lack of ICAM-1 on APCs, but not on T cells, leads to poor T cell activation and proliferation in vitro and in vivo, and that the defect can be compensated by Ag dose, exogenous IL-2, additional costimulation, and by increasing responder T cell density on APCs. ICAM-1-null mice do not respond to immunization with OVA peptide, but immunization with OVA or with Salmonella typhimurium leads to good T cell proliferation 7-10 days later, and clearance of a challenge infection is equivalent to that of wild-type mice. However, when followed over time, recall proliferation and antibacterial immunity decay rapidly in ICAM-1-null mice, while recall cytokine responses are unaffected. The decline in immunity is not related to poor survival of T cells activated on ICAM-1-null APCs, or to poor generation of effectors in ICAM-1-null mice. Phenotypic analysis of T cells stimulated on ICAM-1-null APCs reveals preferential generation of CD44(high) CD62L(low) effector memory cells (T(EM)) over CD44(high) CD62L(high) central memory cells (T(CM)). Further, while the proportion of naive:memory T cells is similar in unmanipulated wild-type and ICAM-1-null mice, there is an accumulation of T(EM) cells, and a high T(EM):T(CM) ratio in aging ICAM-1-null mice. Together, the data indicate that signaling through LFA-1 during T cell activation may be involved in commitment to a proliferation-competent memory pool.     

CXCL12 is a costimulator for CD4+ T cell activation and proliferation in chronic lymphocytic leukemia patients

Activated T cells from patients with chronic lymphocytic leukemia (CLL) provide survival and proliferative signals to the leukemic clone within lymphoid tissues. Recruitment of both, CLL cells and T lymphocytes, to this supportive microenvironment greatly depends on CXCL12 production by stromal and myeloid cells. CXCL12 also supplies survival stimuli to leukemic B cells, but whether it exerts stimulatory effects on T lymphocytes from CLL patients is unknown. In order to evaluate the capacity of CXCL12 to increase CD4⁺ T cell activation and proliferation in CLL patients, peripheral blood mononuclear cells were cultured with or without recombinant human CXCL12 or autologous nurse-like cells, and then T cell activation was induced by anti-CD3 mAb. CXCL12 increases the proliferation and the expression of CD25, CD69, CD154, and IFNγ on CD3-stimulated CD4⁺ T cells from CLL patients, similarly in T cells from ZAP-70⁺ to ZAP-70^? patients. Autologous nurse-like cells establish a close contact with CD4⁺ T cells and increase their activation and proliferation partially through a CXCR4-dependent mechanism. In addition, we found that activated T cells in the presence of CXCL12 enhance the activation and proliferation of the leukemic clone. In conclusion, CXCL12 production by lymphoid tissue microenvironment in CLL patients might play a key dual role on T cell physiology, functioning not only as a chemoattractant but also as a costimulatory factor for activated T cells.     

Ulmus davidiana var. japonica Nakai Upregulates Eosinophils and Suppresses Th1 and Th17 Cells in the Small Intestine

The bark of Ulmus davidiana var. japonica Nakai (Ulmaceae) has been used in traditional Korean medicine for chronic inflammation in the gastrointestinal tract. Here we investigated the frequency and cytokine profile of the major immune cells in the small intestinal lamina propria (SI LP), spleen, and mesenteric lymph nodes (MLNs) of mice treated orally with Ulmus davidiana var. japonica Nakai bark water extract (UDE) to address the immunomodulatory role of this herb in intestinal homeostasis. B6 mice were given 5g/kg UDE once daily for 14 days. They were then sacrificed, and cells were isolated from the spleen, MLNs, and SI LP. The proportion of B versus T lymphocytes, CD4⁺ versus CD8⁺ T lymphocytes, Th1 and Th17 cells, and Foxp3⁺ regulatory T cells in the spleen, MLNs, and SI LP were analyzed. The frequency of antigen-presenting cells (APCs), including dendritic cells, macrophages, and eosinophils in the SI LP and the expression of costimulatory molecules on APCs were also evaluated. The numbers and frequencies of Th1 and Th17 cells in the SI LP were significantly reduced in the UDE-treated mice compared with PBS controls. In addition, the proportion of IL-4-producing eosinophils in the SI LP was significantly elevated in the UDE-treated mice compared with controls. Taken together, these data indicate that UDE up-regulates the number and frequency of SI LP eosinophils, which can down-regulate the Th1 and Th17 responses via IL-4 secretion and contribute to intestinal homeostasis.     

Function and dysfunction of CD4+ T cells in the immune response to melanoma

A major difficulty for tumor immunotherapy derives from the phenomenon that the encounter of the immune system with an antigen does not necessarily result in activation, but may also be followed by the induction of tolerance either by anergy or physical deletion. It is well established that the immune system becomes alerted only in the face of danger, i.e. upon ligand recognition in the context of increased expression of costimulatory molecules, adhesion molecules, and MHC molecules on antigen-presenting cells (APC). The pivotal role of CD4⁺ T lymphocytes in this process has been established. However, encounter of CD4⁺ T cells with either MHC class II-expressing melanoma cells or certain tumor antigen-presenting APC has been reported to induce antigen-specific tolerance. Thus, as more is learned about the molecular regulation of immune responses and the role of CD4⁺ T cells in particular, additional strategies to block inhibitory pathways of T-cell activation will be developed. Such strategies are likely to be based on a modulation of the context in which antigen is encountered by the immune system, e.g. in situ cytokine therapy, induction of costimulatory molecules or the simulation of `danger' signals. Received: 20 March 1999 / Accepted: 3 May 1999     

Signaling pathways activated by leukocyte function-associated Ag-1-dependent costimulation

LFA-1 binding to ICAM-1 can enhance TCR-dependent proliferation of T cells, but it has been difficult to distinguish contributions from increased adhesion, and thus TCR occupancy, versus costimulatory signaling. Whether LFA-1 ligation results in generation of a unique costimulatory signal(s) distinct from those activated by the TCR has been unclear. Using purified ligands, it is shown that ICAM-1 and B7. 1 provide comparable costimulation for proliferation of CD8+ T cells, and that both ligands up-regulate the activities of phosphatidylinositol 3-kinase, sphingomyelinase, and c-Jun NH2-terminal kinase (JNK). These pathways are distinct from those activated by the TCR, and have previously been implicated in up-regulating IL-2 production in response to CD28-B7 interaction. Thus, under conditions in which ICAM-1 provides costimulation of proliferation, LFA-1 ligation activates some of the same signaling pathways as does CD28 ligation. LFA-1 and CD28 do not act identically, however, as indicated by differential sensitivity to inhibitors of phosphatidylinositol 3-kinase; LFA-1-dependent costimulation of proliferation is inhibited, while CD28-dependent costimulation is not. Given the broad distribution of class I and ICAMs on many cell types, the ability of LFA-1 to provide costimulatory signals has implications for where and how CD8+CTL may become activated in response to an antigenic challenge.     

Targeting NKT cells and PD-L1 pathway results in augmented anti-tumor responses in a melanoma model

Invariant or Type 1 NKT cells (iNKT cells) are a unique population of lymphocytes that share characteristics of T cells and natural killer (NK) cells. Various studies have shown that positive costimulatory pathways such as the CD28 and CD40 pathways can influence the expansion and cytokine production by iNKT cells. However, little is understood about the regulation of iNKT cells by negative costimulatory pathways. Here, we show that in vivo activation with α-GalCer results in increased cytokine production and expansion of iNKT cells in the absence of programmed cell death ligand-1 (PD-L1, B7-H1, and CD274). To study whether PD-L1 deficiency on NKT cells would enhance antigen-specific T-cell responses, we utilized CD8⁺ OT-1 OVA transgenic T cells. α-GalCer enhanced the expansion and cytokine production of OT-1 CD8⁺ cells after adoptive transfer into wild-type recipients. However, this expansion was significantly enhanced when OT-1 CD8⁺ T cells were adoptively transferred into PD-L1^−/− recipients. To extend these results to a tumor model, we used the B16 melanoma system. PD-L1^−/− mice given dendritic cells loaded with antigen and α-GalCer had a significant reduction in tumor growth and this was associated with increased trafficking of antigen-presenting cells and CD8⁺ T cells to the tumors. These data demonstrate that abrogating PDL1:PD-1 interactions during the activation of iNKT cells amplifies an anti-tumor response when coupled with DC vaccination.     

The Immunosuppressant Protosappanin A Promotes Dendritic Cell-Mediated Expansion of Alloantigen-Specific Tregs and Prolongs Allograft Survival in Rats

Protosappanin A (PrA), an immunosuppressive ingredient of the medicinal herb Caesalpinia sappan L, prolongs heart allograft survival in rats, possibly by impairing the function of antigen-presenting cells (APCs). We examined the effects of PrA on the maturation and function of dendritic cells (DCs), a potent class of APCs, and the downstream cell–cell and intracellular signaling pathways mediating the immunosuppressive activity of PrA. PrA inhibited LPS-stimulated maturation of Wistar rat DCs in vitro as reflected by reduced expression of costimulatory molecules (CD80 and CD86) and reduced expression of TLR4 and NF-κB, two critical signaling components for antigen recognition. PrA also enhanced the release of IL-10 and decreased the release of IL-12 from DCs, but had no effect on the production of TGF-ß. In mixed cultures, Wistar DCs pretreated with PrA impaired the proliferation of Sprague Dawley (SD) rat T cells while promoting the expansion of SD rat CD4⁺CD25⁺ regulatory T cells (Tregs). Both oral PrA treatment and infusion of PrA-pretreated Wistar DCs prolonged cardiac allograft survival (Wistar donor, SD recipient) and expanded recipient CD4⁺CD25⁺Foxp3⁺ Tregs. Donor spleen cells, but not spleen cells from a third rat strain (DA), supported the expansion of recipient CD4⁺CD25⁺Foxp3⁺ Tregs and suppressed recipient T cell proliferation. We conclude that PrA triggers a tolerogenic state in DCs that allows for the induction of alloantigen-specific Tregs and the suppression of allograft rejection in vivo.     

Nuclear shelter: The influence of subcellular location on the processing of antigens by macroautophagy

CD4⁺ T cells recognize peptides displayed by MHC II molecules on the surface of specialized antigen-presenting cells (APCs). These peptides are generally considered to come from exogenous proteins that have been internalized by APCs and degraded in the endocytic network, thus representing a snapshot of proteins in the APC's extracellular environment. However, it is increasingly apparent that some proteins endogenously expressed within the APC itself can directly gain access to the MHC II pathway. This has particular implications for pathogens that naturally infect APCs, since such infections might therefore become visible to CD4⁺ (MHC II-restricted) effector T cells. Such a pathogen is Epstein-Barr Virus (EBV), a human herpesvirus that infects most individuals establishing life-long infection of B cells. EBV's persistence in the immune host depends upon expression of the viral nuclear protein EBNA1 to segregate the viral genome into daughter cells during homeostatic B cell turnover. Surprisingly, EBNA1 contains many CD4 epitopes, elicits strong CD4 T cell responses in most people and, in in vitro models, can be processed for CD4⁺ T cell recognition through delivery into the MHC II pathway, apparently via macroautophagy (hereafter referred to as autophagy). Taken together, these observations raise the interesting question of how does EBV persist in the B cell system given the presence of strong CD4 T cell responses directed against the one protein the virus absolutely requires for long-term persistence?     

Potent costimulation of human CD8 T cells by anti-4-1BB and anti-CD28 on synthetic artificial antigen presenting cells

The in vitro generation of cytotoxic T lymphocytes (CTLs) for anticancer immunotherapy is a promising approach to take patient-specific therapy from the bench to the bedside. Two criteria must be met by protocols for the expansion of CTLs: high yield of functional cells and suitability for good manufacturing practice (GMP). The antigen presenting cells (APCs) used to expand the CTLs are the key to achieving both targets but they pose a challenge: Unspecific stimulation is not feasible because only memory T cells are expanded and not rare naïve CTL precursors; in addition, antigen-specific stimulation by cell-based APCs is cumbersome and problematic in a clinical setting. However, synthetic artificial APCs which can be loaded reproducibly with MHC-peptide monomers and antibodies specific for costimulatory molecules could resolve these problems. The purpose of this study was to investigate the potential of complex synthetic artificial APCs in triggering the costimulatory molecules CD28 and 4-1BB on the T cell. Anti-4-1BB antibodies were added to an established system of microbeads coated with MHC-peptide monomers and anti-CD28. Triggering via CD28 and 4-1BB resulted in strong costimulatory synergy. The quantitative ratio between these signals determined the outcome of the stimulation with optimal results when anti-4-1BB and anti-CD28 were applied in a 3:1 ratio. Functional CTLs of an effector memory subtype (CD45RA^? CCR7^?) were generated in high numbers. We present a highly defined APC platform using off-the-shelf reagents for the convenient generation of large numbers of antigen-specific CTLs.     

Immature dendritic cells (DCs) use chemokines and intercellular adhesion molecule (ICAM)-1, but not DC-specific ICAM-3-grabbing nonintegrin, to stimulate CD4+ T cells in the absence of exogenous antigen

Dendritic cells (DCs) possess a number of unique features that distinguish them from other APCs. One such feature is their ability to trigger Ag-independent responses in T cells. Previous studies have focused on mature DCs, but the prevalence of this phenomenon in the resting-state immature DCs has never been considered. In this study, we show that, in the absence of Ag, human immature DCs trigger multiple responses in autologous primary CD4+ T cells, namely, increased motility, small Ca2+ transients, and up-regulation of CD69. These responses are particularly marked in CD4+ memory T cells. By using several experimental approaches, we found that DC-specific ICAM-3-grabbing nonintegrin plays no role in the induction of T cell responses, whereas ICAM-1/LFA-1 interactions are required. In addition, DC-produced chemokines contribute to the Ag-independent T cell stimulatory ability of DCs, because pertussis toxin-treated T cells exhibit diminished responses to immature DCs. More particularly, CCL17 and CCL22, which are constitutively produced by immature DCs, mediate both T cell polarization and attraction. Thus, immature DCs owe part of their outstanding Ag-independent T cell stimulatory ability to chemokines and ICAM-1, but not DC-specific ICAM-3-grabbing nonintegrin.     

ICAM-3 interacts with LFA-1 and regulates the LFA-1/ICAM-1 cell adhesion pathway

The interaction of lymphocyte function-associated antigen-1 (LFA-1) with its ligands mediates multiple cell adhesion processes of capital importance during immune responses. We have obtained three anti-ICAM-3 mAbs which recognize two different epitopes (A and B) on the intercellular adhesion molecule-3 (ICAM-3) as demonstrated by sequential immunoprecipitation and cross-competitive mAb-binding experiments. Immunoaffinity purified ICAM-3-coated surfaces were able to support T lymphoblast attachment upon cell stimulation with both phorbol esters and cross-linked CD3, as well as by mAb engagement of the LFA-1 molecule with the activating anti-LFA-1 NKI-L16 mAb. T cell adhesion to purified ICAM-3 was completely inhibited by cell pretreatment with mAbs to the LFA-1 alpha (CD11a) or the LFA-beta (CD18) integrin chains. Anti-ICAM-3 mAbs specific for epitope A, but not those specific for epitope B, were able to trigger T lymphoblast homotypic aggregation. ICAM-3-mediated cell aggregation was dependent on the LFA-1/ICAM-1 pathway as demonstrated by blocking experiments with mAbs specific for the LFA-1 and ICAM-1 molecules. Furthermore, immunofluorescence studies on ICAM-3-induced cell aggregates revealed that both LFA-1 and ICAM-1 were mainly located at intercellular boundaries. ICAM-3 was located at cellular uropods, which in small aggregates appeared to be implicated in cell-cell contacts, whereas in large aggregates it appeared to be excluded from cell-cell contact areas. Experiments of T cell adhesion to a chimeric ICAM-1-Fc molecule revealed that the proaggregatory anti-ICAM-3 HP2/19 mAb was able to increase T lymphoblast attachment to ICAM-1, suggesting that T cell aggregation induced by this mAb could be mediated by increasing the avidity of LFA-1 for ICAM-1. Moreover, the HP2/19 mAb was costimulatory with anti-CD3 mAb for T lymphocyte proliferation, indicating that enhancement of T cell activation could be involved in ICAM-3-mediated adhesive phenomena. Altogether, our results indicate that ICAM-3 has a regulatory role on the LFA-1/ICAM-1 pathway of intercellular adhesion.     

Transfection of the mouse ICAM-1 gene into murine neuroblastoma enhances susceptibility to lysis,reduces in vivo tumorigenicity and decreases ICAM-2-dependent killing

We determined the expression of intercellular adhesion molecules (ICAM) on neuro-2a cells in order to evaluate whether they were involved in cytolysis of murine neuroblastoma. Fluorescence-activated cell sorting analysis revealed that the control neomycin-resistance-genetransduced line (neuro-2a/LN) had poor expression of ICAM-1 (mean channel fluorescence, MCF=3.7). An ICAM-1-positive transfectant of neuro-2a (neuro-2a/ICAM-1⁺) (CMF=64.3) was generated to evaluate directly the role of this adhesion molecule in cytolysis. Neuro-2a/ICAM-1⁺ was more sensitive to LAK killing (69.7% at an effector-to-target ratio of 1001) compared to neuro-2a/LN (48.6%) (P<0.001). Blocking of neuro-2a/LN and neuro-2a/ICAM-1⁺ lysis with anti-ICAM-1 monoclonal antibodies (mAbs) did not account for all the LFA-1-dependent killing. These data indicate that even in neuro-2a/ICAM-1⁺ cells, other LFA-1 ligands participated in the effector-target interaction. Therefore, we examined these cell lines for ICAM-2 expression. Both neuro-2a/LN and neuro-2a/ICAM-1⁺ lines expressed ICAM-2 (MCF=16.4 and 16.5). ICAM-2 accounted for the majority of the LFA-1-dependent killing in the ICAM-1-negative target, neuro-2a/LN, while ICAM-1 played a primary role in the cytolysis of the ICAM-1⁺ transfectant. Inhibition of lysis in the presence of anti-ICAM-1 and ICAM-2 mAbs was comparable to that seen with the addition of anti-LFA-1 mAb, indicating that other LFA-1 ligands were not involved in this system. ICAM-1 expression was associated with decreased in vivo tumorigenicity; mice inoculated with neuro-2a/ICAM-1⁺ cells had a significantly longer survival compared to those receiving neuro-2a/LN cells (median survival time 35.5 versus 24.5 days) (P<0.001). It is important to note that ICAM-1 transfection of murine neuroblastoma did not alter its metastatic potential. We conclude that transfection of mouse neuroblastome with ICAM-1 increases its sensitivity to in vitro lysis and reduces its in vivo tumorgenicity. In ICAM-1-negative murine neuroblastoma cells, ICAM-2 plays a primary role in cell-mediated lysis.This work was supported in part by the Children's Cancer Research Fund, the Minnesota Medical Foundation, the Viking Children's Fund and NIH grants PO1-CA-21737, NO1-AI-85002. E. K. is a recipient of the Irvine McQuarrie Research Scholar Award and B. R. B. a recipient of the Edward Mallinkrodt Foundation Scholar Award