CXCR4-dependent infection of CD8+, but not CD4+, lymphocytes by a primary human immunodeficiency virus type 1 isolate

We recently isolated from an infant an X4-syncytium-inducing (SI) human immunodeficiency virus type 1 (HIV-1) variant (92US143-T8) that was able to infect CD8+ lymphocytes independently of CD4. Although it was CD4 independent, the 92US143-T8 isolate also maintained the ability to infect CD4+ cells. In the present study, we investigated the role of CXCR4 in the infection of CD4+ and CD8+ cells by this primary isolate. The expression of CXCR4 was down modulated in CD8+ lymphocytes after infection with the 93US143-T8 isolate. Infection of CD8+ lymphocytes by the 93US143-T8 isolate was prevented by treatment with AMD3100, a specific antagonist for CXCR4, indicating CXCR4-dependent infection. Interestingly, AMD3100 treatment had no inhibitory role in the infection of purified CD4+ lymphocytes by the same isolate. Furthermore, AMD3100 treatment failed to prevent infection of known CD4+ CXCR4+ T-cell lines (MT-2 and CEM) by the 93US143-T8 isolate. In fact, virus replication in the CD4+ cells was often enhanced in the presence of AMD3100. Viruses produced from the infected CD4+ cells in the presence of AMD3100 maintained an unchanged envelope genotype and an SI phenotype. For the first time, these results provide evidence of CXCR4-dependent infection of CD8+ lymphocytes by a primary HIV-1 isolate. This study also shows a different mode of infection for the CD4+ and CD8+ lymphocytes by the same HIV-1 variant. Finally, our findings suggest that a more careful evaluation is necessary before the random use of AMD3100 as a new entry inhibitor in patients harboring SI HIV-1 strains.     

Regulation of extracellular signal-regulated kinase activity by p120 RasGAP does not involve its pleckstrin homology or calcium-dependent lipid binding domains but does require these domains to regulate cell proliferation.

The gene encoding for p120 RasGAP, has been disrupted in mice (M. Henkemeyer et al., Nature (Lond.), 377: 695-701, 1995).In this study, using fibroblasts derived from these mouse embryos (Gap-/-; P. van der Geer et al., Mol. Cell Biol., 17: 1840-1847, 1997), we demonstrate that mitogen-activated protein kinase (MAPK) activation is prolonged after epidermal growth factor (EGF), but not lysophosphatidic acid, stimulation as compared with wild-type cells. Furthermore, these cells exhibited a moderate increase in their proliferative rate and saturation density, as well as a limited ability to form colonies in soft agar. Stable cell lines expressing full-length p120GAP not only restored the ability to down-regulate MAPK after EGF stimulation but also lowered their saturation densities. Similarly, expression of p120GAP, missing either its pleckstrin homology (PH) or its calcium-dependent lipid binding (CaLB)/C2 domain, restored MAPK down-regulation and retained the ability to associate with p190 RhoGAP and to be phosphorylated by v-src but exhibited higher saturation densities similar to Gap-/- cells. Our results, therefore, suggest that p120GAP functions not only by down-regulating the Ras/MAPK pathway after growth factor stimulation but is also important in regulating cell proliferation that involves its PH and CaLB domains.     

p73 regulates DRAM-independent autophagy that does not contribute to programmed cell death

Evading programmed cell death is a common event in tumour development. The p53 family member, p73, is a potent inducer of death and a determinant of chemotherapeutic response, but different to p53, is rarely mutated in cancer. Understanding cell death pathways downstream of p53 and p73 is therefore pivotal to understand both the development and treatment of malignant disease. Recently, p53 has been shown to modulate autophagy--a membrane trafficking process, which degrades long-lived proteins and organelles. This requires a p53 target gene, DRAM, and both DRAM and autophagy are critical for p53-mediated death. We report here that TA-p73 also regulates DRAM and autophagy, with different TA-p73 isoforms regulating DRAM and autophagy to varying extents. RNAi knockdown of DRAM, however, revealed that p73's modulation of autophagy is DRAM-independent. Also, p73's ability to induce death, again different to p53, is neither dependent on DRAM nor autophagy. In contrast to TA-p73, deltaN-p73 is a negative regulator of p53-induced and p73-induced autophagy, but does not affect autophagy induced by amino-acid starvation. These studies, therefore, represent not only the first report that p73 modulates autophagy but also highlight important differences in the mechanism by which starvation, p53 and p73 regulate autophagy and how this contributes to programmed cell death.     

PGV04, an HIV-1 gp120 CD4 binding site antibody, is broad and potent in neutralization but does not induce conformational changes characteristic of CD4

Recently, several broadly neutralizing monoclonal antibodies (bnMAbs) directed to the CD4-binding site (CD4bs) of gp120 have been isolated from HIV-1-positive donors. These include VRC01, 3BNC117, and NIH45-46, all of which are capable of neutralizing about 90% of circulating HIV-1 isolates and all of which induce conformational changes in the HIV-1 gp120 monomer similar to those induced by the CD4 receptor. In this study, we characterize PGV04 (also known as VRC-PG04), a MAb with potency and breadth that rivals those of the prototypic VRC01 and 3BNC117. When screened on a large panel of viruses, the neutralizing profile of PGV04 was distinct from those of CD4, b12, and VRC01. Furthermore, the ability of PGV04 to neutralize pseudovirus containing single alanine substitutions exhibited a pattern distinct from those of the other CD4bs MAbs. In particular, substitutions D279A, I420A, and I423A were found to abrogate PGV04 neutralization. In contrast to VRC01, PGV04 did not enhance the binding of 17b or X5 to their epitopes (the CD4-induced [CD4i] site) in the coreceptor region on the gp120 monomer. Furthermore, in contrast to CD4, none of the anti-CD4bs MAbs induced the expression of the 17b epitope on cell surface-expressed cleaved Env trimers. We conclude that potent CD4bs bnMAbs can display differences in the way they recognize and access the CD4bs and that mimicry of CD4, as assessed by inducing conformational changes in monomeric gp120 that lead to enhanced exposure of the CD4i site, is not uniquely correlated with effective neutralization at the site of CD4 binding on HIV-1.     

Antibody responses to glycolipid-borne carbohydrates require CD4+ T cells but not CD1 or NKT cells

Naturally occurring anti-carbohydrate antibodies play a major role in both the innate and adaptive immune responses. To elicit an anti-carbohydrate immune response, glycoproteins can be processed to glycopeptides and presented by the classical antigen-presenting molecules, major histocompatibility complex (MHC) Class I and II. In contrast, much less is known about the mechanism(s) for anti-carbohydrate responses to glycolipids, although it is generally considered that the CD1 family of cell surface proteins presents glycolipids to T cells or natural killer T (NKT) cells. Using model carbohydrate systems (isogloboside 3 and B blood group antigen), we examined the anti-carbohydrate response on glycolipids using both antibody neutralisation and knockout mouse-based experiments. These studies showed that CD4(+) T cells were required to generate antibodies to the carbohydrates expressed on glycolipids, and unexpectedly, these antibody responses were CD1d and NKT cell independent. They also did not require peptide help. These data provide new insight into glycolipid antigen recognition by the immune system and indicate the existence of a previously unrecognised population of glycolipid antigen-specific, CD1-independent, CD4(+) T cells.     

In vitro binding and internalization of HIV envelope glycoproteins by human epidermal langerhans cells does not require the CD4-GP120-binding site

Langerhans cells (LC) are epidermal dendritic cells which express several surface antigens, among them the CD4 antigens. Recent data demonstrated that LC constitute target and storage cells for HIV. To better understand the interactions between HIV and LC, we investigated, in the present work, the fate of HIV envelope glycoproteins (gp120 and gp160) incubated with healthy human trypsinized LC in suspensions.After trypsin treatment, only the epitope for OKT4 appeared to be resistant on LC. In the absence of antigenic sites identified by OKT4A, Leu3a or BL4 (epitopes implicated in HIV binding), LC bound and internalized recombinant HIV gp120 or gp160.This finding supports the hypothesis that there exists at the surface of LC a second molecule which may act as an HIV receptor.     

In contrast to effector T cells, CD4+CD25+FoxP3+ regulatory T cells are highly susceptible to CD95 ligand- but not to TCR-mediated cell death

CD4(+)CD25(+)FoxP3(+) regulatory T cells (T(reg)) suppress T cell function and protect rodents from autoimmune disease. Regulation of T(reg) during an immune response is of major importance. Enhanced survival of T(reg) is beneficial in autoimmune disease, whereas increased depletion by apoptosis is advantageous in cancer. We show here that freshly isolated FACS-sorted T(reg) are highly sensitive toward CD95-mediated apoptosis, whereas other T cell populations are resistant to CD95-induced apoptosis shortly after isolation. In contrast, TCR restimulation of T(reg) in vitro revealed a reduced sensitivity toward activation-induced cell death compared with CD4(+)CD25(-) T cells. Thus, the apoptosis phenotype of T(reg) is unique in comparison to other T cells, and this might be further explored for novel therapeutic modulations of T(reg).     

T cell-associated CD18 but not CD62L, ICAM-1, or PSGL-1 is required for the induction of chronic colitis

The induction and perpetuation of chronic colitis are thought to involve a complex set of adhesive interactions between T cells and endothelial cells located on the vasculature within secondary lymphoid tissue and the intestine. The objective of this study was to assess the roles of T cell-associated CD18, CD62L (L-selectin), ICAM-1, and P-selectin glycoprotein ligand-1 (PSGL-1) in the induction of chronic colitis in mice. CD4(+)CD25(-) T cells derived from either wild-type (WT), CD18-deficient [CD18 knockout (KO)], CD62L KO, ICAM-1 KO, or PSGL-1 KO mice were adoptively transferred into recombinase activating gene-1 (RAG-1)-deficient mice (RAG KO mice) to assess the potential of these T cells to induce chronic colitis. At 8-10 wk following T cell transfer, we observed moderate to severe colitis as assessed by increases in colon weight-to-length ratios and by blinded histopathological analysis. In contrast, we found that transfer of CD18 KO T cells into RAG KO recipients resulted in the significant attenuation of colonic inflammation in these mice. Furthermore, we observed fewer infiltrating CD4(+) T cells in the colonic lamina propria in the CD18 KO-->RAG KO group compared with the WT-->RAG KO group. Finally, message levels of colonic TNF-alpha, IL-1beta, and IFN-gamma were significantly reduced in CD18 KO-->RAG KO mice compared with colitic control animals. We conclude that T cell-associated CD18, but not CD62L, ICAM-1, or PSGL-1, is required for the development of chronic colitis.     

Activation of p38 mitogen-activated protein kinase in vivo selectively induces apoptosis of CD8(+) but not CD4(+) T cells

CD4(+) and CD8(+) T cells play specific roles during an immune response. Different molecular mechanisms could regulate the proliferation, death, and effector functions of these two subsets of T cells. The p38 mitogen-activated protein (MAP) kinase pathway is induced by cytokines and environmental stress and has been associated with cell death and cytokine expression. Here we report that activation of the p38 MAP kinase pathway in vivo causes a selective loss of CD8(+) T cells due to the induction of apoptosis. In contrast, activation of p38 MAP kinase does not induce CD4(+) T-cell death. The apoptosis of CD8(+) T cells is associated with decreased expression of the antiapoptotic protein Bcl-2. Regulation of the p38 MAP kinase pathway in T cells is therefore essential for the maintenance of CD4/CD8 homeostasis in the peripheral immune system. Unlike cell death, gamma interferon production is regulated by the p38 MAP kinase pathway in both CD4(+) and CD8(+) T cells. Thus, specific aspects of CD4(+) and CD8(+) T-cell function are differentially controlled by the p38 MAP kinase signaling pathway.     

Insulin-induced p21ras activation does not require protein kinase C, but a protein sensitive to phenylarsine oxide

Insulin treatment of fibroblasts overexpressing the insulin receptor causes a rapid accumulation of the GTP-bound form of p21ras. We have studied the involvement of protein kinase C (PKC) in, and the effect of phenylarsine oxide (PAO), a putative inhibitor of tyrosine phosphatase activity on, this process. Activation of p21ras was not observed when the cells were stimulated with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and pretreatment with TPA for 16 h, sufficient to down-regulate PKC activity, did not abolish p21ras activation by insulin. These results show that PKC is not involved in the insulin-induced activation of p21ras. Pretreatment of the cells with PAO for 5 min completely blocked insulin-induced p21ras activation. Addition of 2,3-dimercaptopropanol prevented this inhibition by PAO. Also, addition of PAO after insulin stimulation could reverse the activation of p21ras. Since PAO did not affect overall phosphorylation of the insulin receptor beta-chain, we conclude that a PAO-sensitive protein is involved in the induction of p21ras activation by insulin.     

Caspase-10-dependent cell death in Fas/CD95 signalling is not abrogated by caspase inhibitor zVAD-fmk

Background

Upon CD95/Fas ligation, the initiator caspase-8 is known to activate effector caspases leading to apoptosis. In the presence of zVAD-fmk, a broad-spectrum caspase inhibitor, Fas engagement can also trigger an alternative, non-apoptotic caspase-independent form of cell death, which is initiated by RIP1. Controversy exists as to the ability of caspase-10 to mediate cell death in response to FasL (CD95L or CD178). Herein, the role of caspase-10 in FasL-induced cell death has been re-evaluated.

Methodology and Principal Findings

The present study shows that FasL-induced cell death was completely impaired in caspase-8- and caspase-10-doubly deficient (I9-2e) Jurkat leukaemia T-cell lines. Over-expressing of either caspase-8 or caspase-10 in I9-2e cells triggered cell death and restored sensitivity to FasL, further arguing for a role of both initiator caspases in Fas apoptotic signalling. In the presence of zVAD-fmk, FasL triggered an alternative form of cell death similarly in wild-type (A3) and in caspase-8-deficient Jurkat cells expressing endogenous caspase-10 (clone I9-2d). Cell death initiated by Fas stimulation in the presence of zVAD-fmk was abrogated in I9-2e cells as well as in HeLa cells, which did not express endogenous caspase-10, indicating that caspase-10 somewhat participates in this alternative form of cell death. Noteworthy, ectopic expression of caspase-10 in I9-2e and HeLa cells restored the ability of FasL to trigger cell death in the presence of zVAD-fmk. As a matter of fact, FasL-triggered caspase-10 processing still occurred in the presence of zVAD-fmk.

Conclusions and Significance

Altogether, these data provide genetic evidence for the involvement of initiator caspase-10 in FasL-induced cell death and indicate that zVAD-fmk does not abrogate caspase-10 processing and cytotoxicity in Fas signalling. Our study also questions the existence of an alternative caspase-independent cell death pathway in Fas signalling.     

Carcinoembryonic antigen-specific but not antiviral CD4+ T cell immunity is impaired in pancreatic carcinoma patients

Pancreatic carcinoma is a very aggressive disease with dismal prognosis. Although evidences for tumor-specific T cell immunity exist, factors related to tumor microenvironment and the presence of immunosuppressive cytokines in patients' sera have been related to its aggressive behavior. Carcinoembryonic Ag (CEA) is overexpressed in 80-90% of pancreatic carcinomas and contains epitopes recognized by CD4(+) T cells. The aim of this study was to evaluate the extent of cancer-immune surveillance and immune suppression in pancreatic carcinoma patients by comparing the anti-CEA and antiviral CD4(+) T cell immunity. CD4(+) T cells from 23 normal donors and 44 patients undergoing surgical resection were tested for recognition of peptides corresponding to CEA and viral naturally processed promiscuous epitopes by proliferation and cytokine release assays. Anti-CEA CD4(+) T cell immunity was present in a significantly higher number of normal donors than pancreatic cancer patients. Importantly, whereas CD4(+) T cells from normal donors produced mainly GM-CSF and IFN-gamma, CD4(+) T cells from the patients produced mainly IL-5, demonstrating a skew toward a Th2 type. On the contrary, the extent of antiviral CD4(+) T cell immunity was comparable between the two groups and showed a Th1 type. The immunohistochemical analysis of tumor-infiltrating lymphocytes showed a significantly higher number of GATA-3(+) compared with T-bet(+) lymphoid cells, supporting a Th2 skew also at the tumor site. Collectively, these results demonstrate that Th2-immune deviation in pancreatic cancer is not generalized but tumor related and suggests that the skew might be possibly due to factor(s) present at the tumor site.     

The HIV-1 gp120 inhibits the binding of adenosine deaminase to CD26 by a mechanism modulated by CD4 and CXCR4 expression

HIV-1 external envelope glycoprotein gp120 inhibits adenosine deaminase (ADA) binding to its cell surface receptor in lymphocytes, CD26, by a mechanism that does not require the gp120-CD4 interaction. To further characterize this mechanism, we studied ADA binding to murine clones stably expressing human CD26 and/or human CD4, and transiently expressing human CXCR4. In this heterologous model, we show that both recombinant gp120 and viral particles from the X4 HIV-1 isolate IIIB inhibited the binding of ADA to wild-type or catalytically inactive forms of CD26. In cells lacking human CXCR4 expression, this gp120-mediated inhibition of ADA binding to human CD26 was completely dependent on the expression of human CD4. In contrast, when cells were transfected with human CXCR4 the inhibitory effect of gp120 was significantly enhanced and was not blocked by anti-CD4 antibodies. These data suggest that the interaction of gp120 with CD4 or CXCR4 is required for efficient inhibition of ADA binding to CD26, although in the presence of CXCR4 the interaction of gp120 with CD4 may be dispensable.     

Protective intestinal anti-rotavirus B cell immunity is dependent on alpha 4 beta 7 integrin expression but does not require IgA antibody production

Rotavirus (RV) is the main cause of severe gastroenteritis in young children; protection has been correlated with intestinal Ab responses. Using a mouse model of RV infection and beta(7)-deficient (beta(7)(-/-)) mice, which do not express alpha(4)beta(7) integrin, we demonstrated the importance of alpha(4)beta(7) integrin in B cell-mediated anti-RV immunity. beta(7)(-/-) mice acutely infected with murine RV resolved infection and developed normal serum IgG Abs but had diminished intestinal IgA responses. alpha(4)beta(7)(-/-) immune B cells did not resolve RV infection when adoptively transferred into RV-infected Rag-2-deficient mice. Fewer RV-specific B cells were found in the intestine of Rag-2-deficient mice transferred with beta(7)(-/-) B cells compared with wild type. The absence of alpha(4)beta(7) expression and/or a lower frequency of IgA-producing cells among transferred beta(7)(-/-) B cells could have accounted for the inability of these cells to resolve RV infection following passive transfer. To distinguish between these possibilities, we studied the importance of IgA production in RV infection using IgA-deficient (IgA(-/-)) mice. IgA(-/-) mice depleted of CD8(+) T cells were able to clear primary RV infection. Similarly, adoptive transfer of immune IgA(-/-) B cells into chronically infected Rag-2-deficient mice resolved RV infection. We further demonstrated in both wild-type and IgA(-/-) mice that, following oral RV infection, protective B cells reside in the alpha(4)beta(7)(high) population. Our findings suggest that alpha(4)beta(7) integrin expression is necessary for B cell-mediated immunity to RV independent of the presence of IgA.     

Fever response to intravenous prostaglandin E2 is mediated by the brain but does not require afferent vagal signaling

PGE2 produced in the periphery triggers the early phase of the febrile response to infection and may contribute to later phases. It can be hypothesized that peripherally synthesized PGE2 transmits febrigenic signals to the brain via vagal afferent nerves. Before testing this hypothesis, we investigated whether the febrigenic effect of intravenously administered PGE2 is mediated by the brain and is not the result of a direct action of PGE2 on thermoeffectors. In anesthetized rats, intravenously injected PGE2 (100 microg/kg) caused an increase in sympathetic discharge to interscapular brown adipose tissue (iBAT), as well as increases in iBAT thermogenesis, end-expired CO2, and colonic temperature (Tc). All these effects were prevented by inhibition of neuronal function in the raphe region of the medulla oblongata using an intra-raphe microinjection of muscimol. We then asked whether the brain-mediated PGE2 fever requires vagal signaling and answered this question by conducting two independent studies in rats. In a study in anesthetized rats, acute bilateral cervical vagotomy did not affect the effects of intravenously injected PGE2 (100 microg/kg) on iBAT sympathetic discharge and Tc. In a study in conscious rats, administration of PGE2 (280 microg/kg) via an indwelling jugular catheter caused tail skin vasoconstriction, tended to increase oxygen consumption, and increased Tc; none of these responses was affected by total truncal subdiaphragmatic vagotomy performed 2 wk before the experiment. We conclude that the febrile response to circulating PGE2 is mediated by the brain, but that it does not require vagal afferent signaling.     

Autoimmunity stimulated by adoptively transferred dendritic cells is initiated by both alphabeta and gammadelta T cells but does not require MyD88 signaling

Vaccination of nonautoimmune prone mice with syngeneic dendritic cells (DC) readily induces anti-DNA autoantibodies but does not trigger systemic disease. We observed that anti-DNA autoantibody generation absolutely required alphabeta T cells and that gammadelta T cells also contributed to the response, but that regulatory T cells restrained autoantibody production. Although both NZB/W F(1) mice and DC vaccinated C57/BL6 mice produced autoantibodies against dsDNA, vaccinated mice had higher levels of Abs against H1 histone and lower levels of antinucleosome Abs than NZB/W F(1) mice. Despite a 100-fold increase in IL-12 and Th1 skewing to a foreign Ag, OVA, synergistic TLR activation of DC in vitro failed to augment anti-DNA Abs or promote class switching beyond that induced by LPS alone. TLR stimulation was not absolutely required for the initial loss of B cell tolerance because anti-DNA levels were similar when wild-type (WT) or MyD88-deficient DC were used for vaccination or WT and MyD88-deficient recipients were vaccinated with WT DC. In contrast, systemic administration of LPS, augmented anti-DNA Ab levels and promoted class switching, and this response was dependent on donor DC signaling via MyD88. LPS also augmented responses in the MyD88-deficient recipients, suggesting that LPS likely exerts its effects on both transferred DC and host B cells in vivo. These results indicate that both the alphabeta and gammadelta subsets are necessary for promoting autoantibody production by DC vaccination, and that although TLR/MyD88 signaling is not absolutely required for initiation, this pathway does promote augmentation, and Th1-mediated skewing, of anti-DNA autoantibodies.     

An epithelial cell line that can stimulate alloproliferation of resting CD4+ T cells, but not after IFN-gamma stimulation

It has previously been shown that IFN-gamma-induced up-regulation of HLA class II on the surface of epithelial cells is not sufficient to induce proliferation of allospecific CD4+ T cells in vitro. To further investigate this phenomenon, a human epithelial bladder carcinoma, T24, was induced to constitutively express HLA class II without IFN-gamma stimulation, by permanent transfection with the full-length class II transactivator (CIITA) gene. Proliferation of allospecific T cells to transfected and wild-type cells with and without prior activation with saturating levels of IFN-gamma for 4 days was examined. IFN-gamma-activated T24 did not induce any response from CD4+ T cells. However, T24.CIITA induced significant levels of alloproliferation, which could be abrogated by pretreatment of T24.CIITA with a mAb to LFA-3. Prestimulation of T24. CIITA with saturating levels of IFN-gamma for 4 days also prevented allospecific CD4+ T cell proliferation. These findings suggest that epithelial cells may be intrinsically able to process and present alloantigen and provide adequate costimulation. We propose that IFN-gamma has a secondary, as yet unidentified, effect that acts to negatively regulate this response, at least in some epithelial cells.     

CD40, but not CD154, expression on B cells is necessary for optimal primary B cell responses

CD40 is an important costimulatory molecule for B cells as well as dendritic cells, monocytes, and other APCs. The ligand for CD40, CD154, is expressed on activated T cells, NK cells, mast cells, basophils, and even activated B cells. Although both CD40(-/-) and CD154(-/-) mice have impaired ability to isotype switch, form germinal centers, make memory B cells, and produce Ab, it is not entirely clear whether these defects are intrinsic to B cells, to other APCs, or to T cells. Using bone marrow chimeric mice, we investigated whether CD40 or CD154 must be expressed on B cells for optimal B cell responses in vivo. We demonstrate that CD40 expression on B cells is required for the generation of germinal centers, isotype switching, and sustained Ab production, even when other APCs express CD40. In contrast, the expression of CD154 on B cells is not required for the generation of germinal centers, isotype switching, or sustained Ab production. In fact, B cell responses are completely normal when CD154 expression is limited exclusively to Ag-specific T cells. These results suggest that the interaction of CD154 expressed by activated CD4 T cells with CD40 expressed by B cells is the primary pathway necessary to achieve B cell activation and differentiation and that CD154 expression on B cells does not noticeably facilitate B cell activation and differentiation.     

Increasing the survival of dendritic cells in vivo does not replace the requirement for CD4+ T cell help during primary CD8+ T cell responses

The survival of dendritic cells (DC) in vivo determines the duration of Ag presentation and is critical in determining the strength and magnitude of the resulting T cell response. We used a mouse model to show that Ag-loaded C57BL/6 DC (MHC class II(+/+) (MHC II(+/+))) that reach the lymph node survived longer than Ag-loaded MHC II(-/-) DC, with the numbers of C57BL/6 DC being approximately 2.5-fold the number of the MHC II(-/-) DC by day 4 and approximately 5-fold by day 7. The differential survival of DC in vivo was not affected by low doses of LPS, but in vitro pretreatment with CD40L or with high doses of LPS increased the numbers of MHC II(-/-) DC to levels approaching those of C57BL/6 DC. Regardless of their numbers and relative survival in lymph nodes, MHC II(-/-) DC were profoundly defective in their ability to induce CTL responses against the gp33 peptide epitope, and were unable to induce expansion and optimal cytotoxic activity of CD8(+) T cells specific for the male Ag UTY. We conclude that CD4(+) T cell help for CD8(+) responses involves mechanisms other than the increased survival of Ag-presenting DC in the lymph node.