VAMP4- and VAMP7-expressing vesicles are both required for cytotoxic granule exocytosis in NK cells

NK cells eliminate cancer and virus-infected cells through their cytolytic activity. The last step in NK-cell cytotoxicity, resulting in exocytosis of granule content, requires fusion of lytic granules with the plasma membrane. Proteins from the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family mediate membrane fusion events in the cell. Here, we show that NK cells express all members of the R-SNARE subgroup. Two of these R-SNARE proteins, VAMP4 and VAMP7, colocalize with lytic granules during cytotoxic interactions. However, only VAMP7 associates with perforin-containing granules in nonactivated cells, indicating that the two VAMPs have different functions in exocytosis. Using both the tumor NK-cell line YTS and the peripheral NK cells, we show that the disruption of expression of either VAMP4 or VAMP7 inhibits the release of lytic granules and severely impairs NK-cell cytotoxic activity. Furthermore, VAMP7 but not VAMP4 is involved in IFN-γ secretion in NK cells, indicating that VAMP7 is involved in many fusion processes and thus plays a more general function in NK-cell activity than VAMP4.     

Calcium influx and signaling in cytotoxic T-lymphocyte lytic granule exocytosis

Summary:  Cytotoxic T lymphocytes (CTLs) kill targets by releasing cytotoxic agents from lytic granules. Killing is a multi-step process. The CTL adheres to a target, allowing its T-cell receptors to recognize antigen. This triggers a signal transduction cascade that leads to the polarization of the microtubule cytoskeleton and granules towards the target, followed by exocytosis that occurs specifically at the site of contact. As with cytokine production by helper T cells (Th cells), target cell killing is absolutely dependent on Ca²⁺ influx, which is involved in regulating both reorientation and release. Current evidence suggests that Ca²⁺ influx in CTLs, as in Th cells, occurs via depletion-activated channels. The molecules that couple increases in Ca²⁺ to reorientation are unknown. The Ca²⁺/calmodulin-dependent phosphatase calcineurin, which plays a critical role in cytokine production by Th cells, is also involved in lytic granule exocytosis, although the relevant substrates remain to be identified and calcineurin activation is only one Ca²⁺ -dependent step involved. There are thus striking similarities and important differences between Ca²⁺ signals in Th cells and CTLs, illustrating how cells can use similar signal transduction pathways to generate different functional outcomes.     

Intravital imaging of CD8<Superscript>+</Superscript> T cell function in cancer

Recent technological advances in photonics are making intravital microscopy (IVM) an increasingly powerful approach for the mechanistic exploration of biological processes in the physiological context of complex native tissue environments. Direct, dynamic and multiparametric visualization of immune cell behavior in living animals at cellular and subcellular resolution has already proved its utility in auditing basic immunological concepts established through conventional approaches and has also generated new hypotheses that can conversely be complemented and refined by traditional experimental methods. The insight that outgrowing tumors must not necessarily have evaded recognition by the adaptive immune system, but can escape rejection by actively inducing a state of immunological tolerance calls for a detailed investigation of the cellular and molecular mechanisms by which the anti-cancer response is subverted. Along with molecular imaging techniques that provide dynamic information at the population level, IVM can be expected to make a critical contribution to this effort by allowing the observation of immune cell behavior in vivo at single cell-resolution. We review here how IVM-based investigation can help to clarify the role of cytotoxic T lymphocytes (CTL) in the immune response against cancer and identify the ways by which their function might be impaired through tolerogenic mechanisms.     

Perforin-dependent cytotoxic mechanism in killing by CD8 positive T cells in ginbuna crucian carp, Carassius auratus langsdorfii

T cell-mediated cytotoxicity occurs via pathways based on perforin or Fas mechanisms. Perforin is a protein present in the cytoplasmic granules of CD8⁺ cytotoxic T lymphocytes and is secreted to form pores on target cell membranes. In fish, although the involvement of perforin in cytotoxicity have been suggested for several species, perforin-mediated cytotoxicity of CD8α⁺ lymphocyte in conjunction with expression of the perforin gene has not been reported. In order to investigate the killing mechanism of CD8α⁺ lymphocytes by perforin-mediated pathway in fish, we measured apoptosis of target cells triggered by CD8α⁺ lymphocytes, performed cytotoxic assays in the presence or absence of perforin inhibitor; concanamycin A and EGTA, and analysed the expression of perforin1, perforin2 and perforin3 isotypic genes in ginbuna crucian carp. In the present study, we found that CTLs attached with target cells. CTL should have direct contact with target cells to kill them. Approximately 50% of target cells were positive for annexin V after co-cultured with CD8α⁺ lymphocytes, indicating the induction of apoptotic cell death. Concanamycin A, which induces depolymerization of perforin resulting in lytic function, suppressed the cytotoxicity of CD8α⁺ cells in a dose-dependent manner. In addition, cytotoxicity mediated by CD8α⁺ lymphocytes were significantly suppressed by the addition of the Ca²⁺-chelating agents EGTA or EGTA-Mg²⁺, and the addition of Ca²⁺ restored the killing mechanism of target cells. We further found enhanced expression of perforin1 but not perforin2 or perforin3 in CTLs from allo-sensitized fish. The present study has demonstrated that ginbuna CTLs kill target cells through perforin-mediated pathway, suggesting that perforin-mediated pathway is conserved throughout vertebrate.     

Diversity of escape variant mutations in Simian virus 40 large tumor antigen (SV40 Tag) epitopes selected by cytotoxic T lymphocyte (CTL) clones

To better understand the relationship between epitope variation and tumor escape from immune surveillance, SV40 T antigen-transformed B6/K-0 cells were subjected to selection with individual CTL clones specific for the SV40 T antigen H-2D(b)-restricted epitopes I or V. CTL-resistant populations were isolated from a majority of the selection cultures and substituted epitope sequences were identified within most of the resistant populations. Tag sequences deleted of all or portions of the selection-targeted epitope were identified, but in lower numbers compared to epitope sequences bearing single residue substitutions. Relatively few flanking residue substitutions were identified, and only in epitope I-targeted selections. The diversity (numbers and epitope residue locations) of substituted epitope residue positions varied between selections. These findings suggest that the scope of spontaneously occurring mutations that could allow for escape from individual CD8+ T cell clones is large.     

In vitro model for the activation of CD4 and CD8 T cell receptors

Previously, most models that sought to explain the misregulation of immune cell function assumed molecular similarities between the disease-causing pathogens and the host's proteins. In recent time several different models have been proposed and in this study, these concepts are compared to a new hypothesis proposing another explanation for this immune dysregulation: the possibility that the mislocalization of proteins may be responsible for autoimmune activity. Based on this hypothesis, proteins are recognized as self or non-self depending on where they appear in sufficiently high concentrations. To examine this new idea, the intracellular human proteins β-actin, GAPDH, and hemoglobin as well as the extracellular human proteins insulin and albumin, were added to human whole blood samples. After an incubation period, the activation of whole-blood T lymphocytes in the samples was measured. The observed activation pattern of the T lymphocytes fit well with the proposed hypothesis. Therefore, these data suggest that protein mislocalization and/or errors within protein trafficking might be important in the development of autoimmune diseases.     

MID2 can substitute for MID1 and control exocytosis of lytic granules in cytotoxic T cells

We have recently shown that the E3 ubiquitin ligase midline 1 (MID1) is upregulated in murine cytotoxic lymphocytes (CTL), where it controls exocytosis of lytic granules and the killing capacity. Accordingly, CTL from MID1 knock‐out (MID1^?/?) mice have a 25–30% reduction in exocytosis of lytic granules and cytotoxicity compared to CTL from wild‐type (WT) mice. We wondered why the MID1 gene knock‐out did not affect exocytosis and cytotoxicity more severely and speculated whether MID2, a close homologue of MID1, might partially compensate for the loss of MID1 in MID1^?/? CTL. Here, we showed that MID2, like MID1, is upregulated in activated murine T cells. Furthermore, MID1^?/? CTL upregulated MID2 two–twenty‐fold stronger than CTL from WT mice, suggesting that MID2 might compensate for MID1. In agreement, transfection of MID2 into MID1^?/? CTL completely rescued exocytosis of lytic granules in MID1^?/? CTL, and vice versa, knock‐down of MID2 inhibited exocytosis of lytic granules in both WT and MID1^?/? CTL, demonstrating that both MID1 and MID2 play a central role in the regulation of granule exocytosis and that functional redundancy exists between MID1 and MID2 in CTL.     

A distinct subset of intestinal dendritic cells responds selectively to oral TLR7/8 stimulation

The intestinal innate immune system continually interacts with commensal bacteria, thus oral vaccines should induce extra/alternative activation of DC, potentially through TLR. To examine this we collected intestinal lymph DC (iL-DC) under steady-state conditions and after feeding resiquimod (R-848), a synthetic TLR7/8 ligand, which we showed induces complete emptying of gut DC into lymph. iL-DC are heterogeneous with subset-specific functions. In this study we determined the kinetics of iL-DC subset release, activation and cytokine secretion induced by R-848. We show that L-DC comprise three distinct subsets (CD172ahigh, CD172aint and CD172alow) present with similar frequencies in intestinal but not hepatic lymph. No iL-DC express TLR7 mRNA, and only CD172a+ iL-DC express TLR8. However, after oral R-848 administration, output of all three subsets increases dramatically. CD172ahigh DC release precedes that of CD172alow DC, and the increased frequency of CD25high iL-DC is restricted to the two CD172a+ subsets. After feeding R-848 only CD172ahigh iL-DC secrete IL-6 and IL-12p40. However, CD172aint and CD172ahigh DC secrete similar but markedly lower amounts when stimulated in vitro. These results highlight the importance of in vivo approaches to assess adjuvant effects on DC and give novel insights into the subset-specific effects of an oral TLR ligand on intestinal DC.     

A role for ARF6 in dendritic cell podosome formation and migration

ADP-ribosylation factor 6 (ARF6) is a widely expressed GTPase that influences both membrane traffic and actin cytoskeleton function. Its role in dendritic cells (DC) has not previously been investigated. We analysed the effect of retroviral expression of ARF6 GDP/GTP binding and other functional mutants in primary murine DC. Maturation in response to lipopolysaccharide (LPS) proceeded normally in DC expressing ARF6 mutants and production of inflammatory cytokines was similarly unaffected. Although LPS-stimulated macropinocytosis was suppressed by expression of the GTP-binding Q67L ARF6 mutant we detected no overall activation of ARF6 by LPS. The ability of immature DC to migrate towards CCL3 and to a lesser extent, of mature DC to migrate towards CCL19, was compromised by expression of either the Q67L or the GDP-binding T44N mutant. Examination of the actin cytoskeleton in these cells revealed that both mutants strongly inhibited the formation of F-actin-rich podosomes, providing a possible explanation for the effects of ARF6 mutants on DC migration. Thus, these studies identify responses in DC that require normal ARF6 function, though not necessarily further ARF6 activation. They reveal for the first time a role for ARF6 in podosome formation and demonstrate functional effects of the T44N ARF6 mutant.     

Requirement for Q226, but not multiple charged residues,in the class I MHC CD loop/D strand for TCR-activated CD8 accessory function

Activation of CD8+ cytotoxic T lymphocytes typically begins with recognition of class I MHC-peptide complexes by the TCR and CD8 as a coreceptor. In its coreceptor role, CD8 binds thesame class I-peptide antigen complex as the TCR, enhancing the strength of TCR-class I interaction. Subsequent to initial TCR engagement, CD8 acts as an accessory molecule by binding any properly conformed class I molecules on the target cell surface, leading to CD8-mediated adhesion and cosignaling functions. We expressed and isolated a number of mutant class I molecules in which one or moreacidic or polar residues in the class I alpha3 domain CD loop and D strand region, or alpha2 domain were altered. Using solid phase CTL adhesion and degranulation assays with isolated class I molecules, we demonstrate that multiple acidic residues in the alpha3 domain, although involved in CD8 coreceptor interaction, are not required for TCR-activated CD8 accessory interactions. Instead, we show that Q226, a polar group on the end of the CD loop, is required for TCR-activated CD8 accessory functions. These results indicate that CD8 coreceptor and accessory interactions differ substantially and suggest that TCR activation results in changes that alter the structural constraints for CD8 accessory interactions.     

Non-redundant role for IL-7R signaling for the survival of CD8+ memory T cells

IL-7 and IL-15 are important cytokines for CD8 memory T cells. However, the extent that IL-7 is essential for CD8 T cell memory remains unclear because blocking IL-7 in vivo results in near complete inhibition of T cell development with the few mature T cells exhibiting functional abnormalities. To bypass this complication, CD8 memory development was examined utilizing a mouse model where transgenic IL-7Ralpha was selectively expressed in the thymus of IL-7Ralpha(-/-) mice. T cell development was corrected but the resulting peripheral T cells were essentially IL-7 non-responsive. Activation of IL-7R-defective OT-I CD8(+) T cells with OVA(257-264) and IL-2 readily yielded CTL. Upon further culture with IL-15, these CTL expressed phenotypic and functional properties of central memory-like cells. Thus, IL-7R-defective CD8(+) T cells do not exhibit intrinsic defects in effector or memory development. When IL-7R-defective OT-I CTL were adoptively transferred into normal or IL-15(-/-) recipient mice in a non-inflammatory setting, they converted into memory-like cells, but did not persist, which was even more striking in IL-15(-/-) recipients. This poor persistence was rescued after expression of transgenic Bcl-2 in IL-7R-defective OT-I T cells. Collectively, these data indicate that IL-7 is non-redundantly required for the survival of CD8 memory T cells.     

Generation of functionally active HIV-1 specific CD8+ CTL in intestinal mucosa following mucosal, systemic or mixed prime-boost immunization

Gastrointestinal and vaginal mucosa are major sites of entry in natural HIV infection and therefore the preferred sites to elicit high-avidity CD8⁺ CTL by vaccination. We directly compare systemic and mucosal immunization in mice after DNA priming and boosting with rgp160 env expressed either in MVA or Ad for their ability to induce mucosal as well as systemic HIV-specific CTL. The optimal CTL response in the gut mucosa was observed after priming with the HIV-1 gp160 env DNA vaccine and boosting with rMVA or rAd encoding the same envelope gene all administered intrarectally (IR). Maximum levels of high-avidity CD8⁺ T cells were seen in intestinal lamina propria following this regimen. When the prime and boost routes were distinct, the delivery site of the boost had a greater impact than the DNA priming. IM DNA prime and IR rMVA boost were more effective than IR DNA prime and IM rMVA boost for eliciting mucosal CD8⁺ T-cell avidity. A systemic DNA-prime-followed by systemic rMVA boost induced high levels of high-avidity CD8⁺ T cells systemically, but responses were undetectable in mucosal sites. A single systemic immunization with rMVA was sufficient to induce high-avidity IFN-γ secreting CD8⁺ T cells in systemic organs, whereas a single mucosal immunization with rMVA was not sufficient to elicit high-avidity CD8⁺ T cells in mucosa. Thus, a heterologous mucosal DNA prime-viral vectored boost strategy was needed. The requirement for a heterologous DNA prime-recombinant viral boost strategy for generation of high-avidity CD8⁺ T cells in mucosal sites in mice may be more stringent than for the induction of high-avidity CD8⁺ T cells in systemic compartments.     

Early detection of dominant Env-specific and subdominant Gag-specific CD8+ lymphocytes in equine infectious anemia virus-infected horses using major histocompatibility complex class I/peptide tetrameric complexes

Cytotoxic T lymphocytes (CTL) are critical for control of lentiviruses, including equine infectious anemia virus (EIAV). Measurement of equine CTL responses has relied on chromium-release assays, which do not allow accurate quantitation. Recently, the equine MHC class I molecule 7-6, associated with the ELA-A1 haplotype, was shown to present both the Gag-GW12 and Env-RW12 EIAV CTL epitopes. In this study, 7-6/Gag-GW12 and 7-6/Env-RW12 MHC class I/peptide tetrameric complexes were constructed and used to analyze Gag-GW12- and Env-RW12-specific CTL responses in two EIAV-infected horses (A2164 and A2171). Gag-GW12 and Env-RW12 tetramer-positive CD8+ cells were identified in nonstimulated peripheral blood mononuclear cells as early as 14 days post-EIAV inoculation, and frequencies of tetramer-positive cells ranged from 0.4% to 6.7% of nonstimulated peripheral blood CD8+ cells during the 127-day study period. Although both horses terminated the initial viremic peak, only horse A2171 effectively controlled viral load. Neutralizing antibody was present during the initial control of viral load in both horses, but the ability to maintain control correlated with Gag-GW12-specific CD8+ cells in A2171. Despite Env-RW12 dominance, Env-RW12 escape viral variants were identified in both horses and there was no correlation between Env-RW12-specific CD8+ cells and control of viral load. Although Gag-GW12 CTL escape did not occur, a Gag-GW12 epitope variant arose in A2164 that was recognized less efficiently than the original epitope. These data indicate that tetramers are useful for identification and quantitation of CTL responses in horses, and suggest that the observed control of EIAV replication and clinical disease was associated with sustained CTL recognition of Gag-specific epitopes.     

The process of granule exocytosis in non-stimulated atrial granular cells of the snail, Achatina achatina: An ultrastructural,histochemical and immunocytochemical study

Abundant secretory granular cells (GCs) in the Giant African land snail atrium harbor a range of bioactive substances and undergo rapid total degranulation in response to stimulation of the cardiac nerve or stressful influences. Here we have analyzed exocytotic events in the non-stimulated GCs. It was shown that the GCs contain three major distinct types of granules that differ histochemically, immunocytochemically and ultrastructurally, each performing specific functions. The type I granules characteristically filled with electron-lucent homogeneous materials exhibit intense immunoreactivity for bioactive proteins and therefore are considered to be storage granules. Histochemistry using vital staining with Acridine Orange and Gomori acid phosphatase technique has revealed lysosomal-related nature of the electron-dense type II granules. Digestion remnants appearing as fine filamentous materials fill the type III granules. Only the type III granules fuse together and with the plasma membrane form degranulation channels and surface pores, through which the debris is removed from the cell. The finding of granules exhibiting intermediate ultrastructural, histochemical and immunocytochemical features suggests that the major granule types represent most stable states along a granule empting continuum. Thus, under physiological conditions, the GCs continuously produce secretory proteins and so maintain readiness for stress-response, but use protein degradation machinery to prevent massive release of these bioactive substances into hemolymph.     

Distinctive role of CCR7 in migration and functional activity of naive- and effector/memory-like Treg subsets

Foxp3+CD25+CD4+ Treg play a fundamental role in the maintenance of self tolerance and the control of inflammatory reactions. Previous data demonstrated a division of labor between naive- and effector/memory-like Treg subsets, which is largely based on their lymph node-recirculating and inflammation-seeking migration behavior, respectively. The chemokine receptor CCR7 is expressed on both types of Treg subsets, albeit at different levels. Whether it fulfills similar or distinct roles in these subsets has not been studied so far. We here show that the recirculation of naive-like Treg through LN and, to some extent, the gut is dependent on CCR7. Lack of CCR7 not only prevents recirculation, but also almost completely abolishes the ability of naive-like Treg to control the priming phase of an immune response. In contrast, CCR7 deficiency in effector/memory-like Treg promotes their accumulation in inflamed sites, compatible with a role of CCR7 for exit from the tissue. Local Treg accumulation was accompanied by an enhanced suppression of inflammation. Together, our findings provide conclusive evidence that CCR7 expression on Treg differentially controls in vivo function of the naive- and effector/memory-like subsets.     

A direct role for Wnt8 in ventrolateral mesoderm patterning

Vertebrate dorsoventral patterning requires both Wnt8 and BMP signaling. Because of their multiple interactions, discerning roles attributable specifically to Wnt8 independent of BMP has been a challenge. For example, Wnt8 represses the dorsal organizer that negatively regulates ventral BMP signals, thus Wnt8 loss‐of‐function phenotypes may reflect the combined effects of reduced Wnt8 and BMP signaling. We have taken a loss‐of‐function approach in the zebrafish to generate embryos lacking expression of both Wnt8 and the BMP antagonist Chordin. wnt8;chordin loss‐of‐function embryos show rescued BMP signaling, thereby allowing us to identify Wnt8‐specific requirements. Our analysis shows that Wnt8 is uniquely required to repress prechordal plate specification but not notochord, and that Wnt8 signaling is not essential for specification of tailbud progenitors but is required for normal expansion of posterior mesoderm cell populations. Thus, Wnt8 and BMP signaling have independent roles during vertebrate ventrolateral mesoderm development that can be identified through loss‐of‐function analysis. Developmental Dynamics 239:2828–2836, 2010. © 2010 Wiley‐Liss, Inc.     

A membrane-located glycosphingolipid of monocyte/granulocyte lineage cells induces growth arrest and triggers the lytic viral cycle in Epstein-Barr virus genome-positive Burkitt lymphoma lines

Gangliosides are known to influence cell growth and differentiation. The neolacto series ganglioside IV³NeuAc-nLc₄ (2→3-sialosylparagloboside) is present in members of the monocyte/granulocyte lineage, but is not found in cells that belong to the lymphocyte lineage. In this study we demonstrated that IV³NeuAc-nLc₄ inhibits the proliferation of Epstein-Barr virus (EBV) genome-positive Burkitt lymphoma cells of the lines Raji and P3HR-1K. IV³NeuAc-nLc₄-induced growth inhibition is associated with an increase in G₀/G₁ phase cells and a reduced expression of CD21 and HLA-DR antigens on Raji cells. These data suggest that IV³NeuAc-nLc₄ may affect differentiation of lymphoma cells. Additionally, the increased expression of viral mRNA species which are characteristic for the lytic viral cycle in the non-producer line Raji and the enhanced release of virions from the producer line P3HR-1K demonstrate that IV³NeuAc-nLc₄ activates the replication of EBV. Growth inhibition and termination of the viral latency suggest that IV³NeuAc-nLc₄ present in monocyte/granulocyte lineage cells may be an effector of the natural defense against EBV persistency and transformation. Received: 20 December 1998     

Differential roles for CXCR3 in CD4+ and CD8+ T cell trafficking following viral infection of the CNS

Lymphocyte infiltration into the central nervous system (CNS) following viral infection represents an important component of host defense and is required for control of viral replication. However, the mechanisms governing inflammation in response to viral infection of the CNS are not well understood. Following intracranial (i.c.) infection of susceptible mice with mouse hepatitis virus (MHV), mice develop an acute encephalomyelitis followed by a chronic demyelinating disease. The CXC chemokine ligand 10 (CXCL10) is expressed following MHV infection and signals T cells to migrate into the CNS. The functional contribution of the CXCL10 receptor CXCR3 in host defense and disease in response to MHV infection was evaluated. The majority of CD4+ and CD8+ T cells infiltrating the CNS following MHV infection express CXCR3. Administration of anti-CXCR3 antibody reduced CD4+ T cell infiltration (p相似文献   

Lymphocyte binding to vascular endothelium in inflamed skin revisited: a central role for vascular adhesion protein-1 (VAP-1)

The binding of leukocytes to vascular endothelium and their migration into tissues is mediated by adhesion molecules on the endothelial cells and leukocytes. Vascular adhesion protein-1 (VAP-1) is a 170–180/90-kDa endothelial molecule expressed most prominently in high endothelial venules in peripheral lymph node (PLN) type lymphatic tissues. VAP-1 mediates lymphocyte binding to PLN, tonsil and synovium. The expression of VAP-1 is induced in inflammatory diseases such as arthritis and gut inflammation. We examined the expression, structure and function of VAP-1 in normal and inflamed skin and compared it to those of other adhesion molecules implicated in skin homing. In psoriasis, lichen ruber planus, pemphigoid and allergic lesions, VAP-1 was markedly up-regulated. The expression of VAP-1 was also increased in biopsies of healthy skin of the patients. The VAP-1 molecule induced in skin is decorated with abundant sialic acids. VAP-1 in inflamed skin is functional, since inhibition with anti-VAP-1 monoclonal antibodies caused a 60% reduction in lymphocyte adhesion to vascular endothelium. Antibodies against E-selectin, which has been regarded as the major vascular addressin directing cutaneous lymphocyte traffic, and, surprisingly, against peripheral lymph node addressin (PNAd), caused inhibitions of 30% and 60%, respectively, in the frozen section adhesion assay. These findings suggest important roles also for VAP-1 and PNAd in lymphocyte homing into inflamed skin.     

Use of herpesvirus saimiri-immortalized macaque CD4(+) T cell clones as stimulators and targets for assessment of CTL responses in macaque/AIDS models

Herpesvirus saimiri (HVS), a nonhuman primate γ herpes virus, was used to immortalize pig-tailed macaque CD4⁺ T lymphocytes. The HVS-immortalized T cell lines were used to develop CD4⁺ T cell clones from two animals. Three CD4⁺ T cell clones were further characterized for the expression of cell surface markers. All expressed CD2, CD4, CD58, CD69 and CD80 and therefore resembled activated T cells. These clones required exogenous IL-2 for efficient growth and were found to be highly susceptible to infection by the challenge virus, Chimeric simian/human immunodeficiency virus (SHIV_KU-1). They could also be productively infected not only by the quasispecies of the challenge virus (SHIV_KU-1/PDJ and SHIV_KU-1/PNA, isolated from macaque PDj and PNa, respectively) but also by a different chimeric simian/human immunodeficiency virus (SHIV_89.6P) and simian immunodeficiency virus (SIV_MAC239). The virus-infected CD4⁺ T cell clones were also used as stimulators for generation of CTL effectors. These effectors exhibited excellent virus-specific lysis in chromium-release assays when syngenic SHIV_KU-1 infected autologous CD4⁺ T cell clones were used as targets. The target cell lysis was virus specific, as uninfected control cells showed no or minimal lysis.