Reduced protection from simian immunodeficiency virus SIVmac251 infection afforded by memory CD8+ T cells induced by vaccination during CD4+ T-cell deficiency

Adaptive CD4⁺ and CD8⁺ T-cell responses have been associated with control of human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) replication. Here, we have designed a study with Indian rhesus macaques to more directly assess the role of CD8 SIV-specific responses in control of viral replication. Macaques were immunized with a DNA prime-modified vaccinia virus Ankara (MVA)-SIV boost regimen under normal conditions or under conditions of antibody-induced CD4⁺ T-cell deficiency. Depletion of CD4⁺ cells was performed in the immunized macaques at the peak of SIV-specific CD4⁺ T-cell responses following the DNA prime dose. A group of naïve macaques was also treated with the anti-CD4 depleting antibody as a control, and an additional group of macaques immunized under normal conditions was depleted of CD8⁺ T cells prior to challenge exposure to SIV_mac251. Analysis of the quality and quantity of vaccine-induced CD8⁺ T cells demonstrated that SIV-specific CD8⁺ T cells generated under conditions of CD4⁺ T-cell deficiency expressed low levels of Bcl-2 and interleukin-2 (IL-2), and plasma virus levels increased over time. Depletion of CD8⁺ T cells prior to challenge exposure abrogated vaccine-induced protection as previously shown. These data support the notion that adaptive CD4⁺ T cells are critical for the generation of effective CD8⁺ T-cell responses to SIV that, in turn, contribute to protection from AIDS. Importantly, they also suggest that long-term protection from disease will be afforded only by T-cell vaccines for HIV that provide a balanced induction of CD4⁺ and CD8⁺ T-cell responses and protect against early depletion of CD4⁺ T cells postinfection.     

Isolation of CD4+ T cells from mouse lymph nodes using Miltenyi MACS purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are > or = 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!     

Differential kinetics and specificity of EBV-specific CD4+ and CD8+ T cells during primary infection

The generation and maintenance of virus-specific CD4(+) T cells in humans are not well understood. We used short in vitro stimulation assays followed by intracellular cytokine staining to characterize the timing, magnitude, and Ag specificity of CD4(+) T cells over the course of primary EBV infection. Lytic and latent protein-specific CD4(+) T cells were readily detected at presentation with acute infectious mononucleosis and declined rapidly thereafter. Responses to BZLF-1, BMLF-1, and Epstein-Barr nuclear Ag-3A were more commonly detected than responses to Epstein-Barr nuclear Ag-1. Concurrent analyses of BZLF-1-specific CD4(+) and CD8(+) T cells revealed differences in the expansion, specificity, and stability of CD4(+) and CD8(+) T cell-mediated responses over time. Peripheral blood EBV load directly correlated with the frequency of EBV-specific CD4(+) T cell responses at presentation and over time, suggesting that EBV-specific CD4(+) T cell responses are Ag-driven.     

Timed ablation of regulatory CD4+ T cells can prevent murine AIDS progression

We describe successful immunotherapy of murine AIDS (MAIDS) in C57BL/6J mice based on the elimination of replicating CD4(+) regulator T cells. We demonstrate that a single injection of the antimitotic drug vinblastine (Vb) given 14 days postinfection (p.i.) with LP-BM5 can prevent MAIDS progression. Treatment with anti-CD4 mAb at 14 days p.i. is similarly able to prevent MAIDS. Treatment at other time points with Vb or anti-CD4 mAb is ineffective. The effect is based on ablation of a replicating dominantly suppressive CD4(+) T cell population, as indicated by adoptive transfer and in vivo depletion experiments using mAbs against CD4 as well as combinations of mAbs against the known regulatory cell surface markers CD25, GITR, and CTLA-4. Cell surface marker analysis shows a population of CD4(+)CD25(+) cells arising shortly before day 14 p.i. Cytokine analyses show a peak in IL-10 production from day 12 to day 16 p.i. MAIDS-infected mice also have CD4(+) T cells with significantly higher expression levels of CD38 and particularly CD69, which have been demonstrated to be regulator T cell markers in the Friend retroviral model. The immunotherapy appears to prevent disease progression, although no protection against reinfection with LP-BM5 is generated. These data define a new therapy for murine retroviral infection, which has potential for use in other diseases where T regulator cell-mediated immunosuppression plays a role in the disease process.     

Dichotomy between CD1a+ and CD83+ dendritic cells in lymph nodes during SIV infection of macaques

The prevalence and differentiation of dendritic cells (DC) in lymphoid tissue of simian immunodeficiency virus (SIV)-infected cynomolgus monkeys was studied during disease progression. Lymph node biopsies were consecutively obtained from clinical rapid and slow progressors until the development of disease consistent with simian acquired immunodeficiency syndrome (sAIDS) occurred. Quantitative evaluation of CD1a+ DC and the expression of DC antigens related to maturation (CD83, DC-LAMP and S100b) were performed at the single cell level by in situ image analysis. Despite a persistent prevalence of CD1a+ DC in lymphoid tissue during disease progression, there was a subsequent drop of mature CD83+, DC-LAMP+ and S100b+ DC, correlating with the decline of CD4+ T cells in blood. Thus, disease progression to sAIDS was associated with impaired maturation of DC, and lack of CD83, DC-LAMP and S100b expression.     

Non-hematopoietic cells in lymph nodes drive memory CD8 T cell inflation during murine cytomegalovirus infection

During human and murine cytomegalovirus (MCMV) infection an exceptionally large virus-specific CD8 T cell pool is maintained in the periphery lifelong. This anomalous response is only seen for specific subsets of MCMV-specific CD8 T cells which are referred to as 'inflationary T cells'. How memory CD8 T cell inflation is induced and maintained is unclear, though their activated phenotype strongly suggests an involvement of persistent antigen encounter during MCMV latency. To dissect the cellular and molecular requirements for memory CD8 T cell inflation, we have generated a transgenic mouse expressing an MHC class I-restricted T cell receptor specific for an immunodominant inflationary epitope of MCMV. Through a series of adoptive transfer experiments we found that memory inflation was completely dependent on antigen presentation by non-hematopoietic cells, which are also the predominant site of MCMV latency. In particular, non-hematopoietic cells selectively induced robust proliferation of inflationary CD8 T cells in lymph nodes, where a majority of the inflationary CD8 T cells exhibit a central-memory phenotype, but not in peripheral tissues, where terminally differentiated inflationary T cells accumulate. These results indicate that continuous restimulation of central memory CD8 T cells in the lymph nodes by infected non-hematopoietic cells ensures the maintenance of a functional effector CD8 T pool in the periphery, providing protection against viral reactivation events.     

Generation of antifungal effector CD8+ T cells in the absence of CD4+ T cells during Cryptococcus neoformans infection

Immunity to the opportunistic fungus Cryptococcus neoformans is dependent on cell-mediated immunity. Individuals with defects in cellular immunity, CD4(+) T cells in particular, are susceptible to infection with this pathogen. In host defense against a number of pathogens, CD8(+) T cell responses are dependent upon CD4(+) T cell help. The goal of these studies was to determine whether CD4(+) T cells are required for the generation of antifungal CD8(+) T cell effectors during pulmonary C. neoformans infection. Using a murine intratracheal infection model, our results demonstrated that CD4(+) T cells were not required for the expansion and trafficking of CD8(+) T cells to the site of infection. CD4(+) T cells were also not required for the generation of IFN-gamma-producing CD8(+) T cell effectors in the lungs. In CD4(-) mice, depletion of CD8(+) T cells resulted in increased intracellular infection of pulmonary macrophages by C. neoformans, increasing the pulmonary burden of the infection. Neutralization of IFN-gamma in CD4(-)CD8(+) mice similarly increased macrophage infection by C. neoformans, thereby blocking the protection provided by CD8(+) T cells. Altogether, these data support the hypothesis that effector CD8(+) T cell function is independent of CD4(+) T cells and that IFN-gamma production from CD8(+) T cells plays a role in controlling C. neoformans by limiting survival of C. neoformans within macrophages.     

Sequential activation of CD8+ T cells in the draining lymph nodes in response to pulmonary virus infection

We have used a TCR-transgenic CD8+ T cell adoptive transfer model to examine the tempo of T cell activation and proliferation in the draining lymph nodes (DLN) in response to respiratory virus infection. The T cell response in the DLN differed for mice infected with different type A influenza strains with the onset of T cell activation/proliferation to the A/JAPAN virus infection preceding the A/PR8 response by 12-24 h. This difference in T cell activation/proliferation correlated with the tempo of accelerated respiratory DC (RDC) migration from the infected lungs to the DLN in response to influenza virus infection, with the migrant RDC responding to the A/JAPAN infection exhibiting a more rapid accumulation in the lymph nodes (i.e., peak migration for A/JAPAN at 18 h, A/PR8 at 24-36 h). Furthermore, in vivo administration of blocking anti-CD62L Ab at various time points before/after infection revealed that the virus-specific CD8+ T cells entered the DLN and activated in a sequential "conveyor belt"-like fashion. These results indicate that the tempo of CD8+ T cell activation/proliferation after viral infection is dependent on the tempo of RDC migration to the DLN and that T cell activation occurs in an ordered sequential fashion.     

CD4+CD25+ regulatory T cells in HIV infection

The immune system faces the difficult task of discerning between foreign, potentially pathogen-derived antigens and self-antigens. Several mechanisms, including deletion of self-reactive T cells in the thymus, have been shown to contribute to the acceptance of self-antigens and the reciprocal reactivity to foreign antigens. Over the last decade it has become increasingly clear that CD4(+)CD25(+) T(Reg) cells are crucial for maintenance of T cell tolerance to self-antigens in the periphery, and to avoid development of autoimmune disorders. Recently, evidence has also emerged that demonstrates that CD4(+)CD25(+) T(Reg) cells can also suppress T cell responses to foreign pathogens, including viruses such as HIV. In this article we review the current knowledge and potential role of CD4(+)CD25(+) T(Reg) cells in HIV infection.     

Dynamics of CD4^＋CD25^＋ T Cells in Spleens and Mesenteric Lymph Nodes of Mice Infected with Schistosomajaponicum

CD4~ CD25~ T cells play a major role in modulating immune response,but few reports havebeen published about schistosomiasis.Here,we investigated the changes in CD4~ CD25~ T cell populations inspleens and mesenteric lymph nodes of mice infected with Schistosoma japonicum.The proportions ofCD4~ CD25~ T cells in total CD4~ T cells were analyzed by flow cytometry.CD25 and Foxp3 expression wasmeasured by real-time quantitative polymerase chain reaction.The suppressive activities of CD4~ CD25~ Tcells were detected by in vitro proliferation of splenocytes.Evidence showed that the percentage of CD4~ CD25~ T cells was the same as controls 3 weeks post-infection.At the acute stage of infection,the percentagedecreased significantly.However,at the chronic stage of infection,it rebounded to normal levels or evenhigher.The expression of the CD25 and Foxp3 showed gradual increase along with the infection progress.Invitro experiment also showed the strong suppressive effect of CD4~ CD25~ T cells,isolated during the chronicstage,on proliferation of the CD25~-splenocytes.This is the first time that the dynamics of CD4~ CD25~ T cellpopulations was demonstrated in mice infected with schistosomiasis.In conclusion,our data indicated thatCD4~ CD25~ cells might be involved in the immune modulation during S.japonicum infection,which en-hances current knowledge of the mechanisms of the immuno-downregulation and re-infection inschistosomiasis.     

CXCR6+CD4+ T cells promote mortality during Trypanosoma brucei infection

Liver macrophages internalize circulating bloodborne parasites. It remains poorly understood how this process affects the fate of the macrophages and T cell responses in the liver. Here, we report that infection by Trypanosoma brucei induced depletion of macrophages in the liver, leading to the repopulation of CXCL16-secreting intrahepatic macrophages, associated with substantial accumulation of CXCR6⁺CD4⁺ T cells in the liver. Interestingly, disruption of CXCR6 signaling did not affect control of the parasitemia, but significantly enhanced the survival of infected mice, associated with reduced inflammation and liver injury. Infected CXCR6 deficient mice displayed a reduced accumulation of CD4⁺ T cells in the liver; adoptive transfer experiments suggested that the reduction of CD4⁺ T cells in the liver was attributed to a cell intrinsic property of CXCR6 deficient CD4⁺ T cells. Importantly, infected CXCR6 deficient mice receiving wild-type CD4⁺ T cells survived significantly shorter than those receiving CXCR6 deficient CD4⁺ T cells, demonstrating that CXCR6⁺CD4⁺ T cells promote the mortality. We conclude that infection of T. brucei leads to depletion and repopulation of liver macrophages, associated with a substantial influx of CXCR6⁺CD4⁺ T cells that mediates mortality.     

Uncoupling of IL-2 signaling from cell cycle progression in naive CD4+ T cells by regulatory CD4+CD25+ T lymphocytes

Prior reports have shown that CD4(+)CD25(+) regulatory T cells suppress naive T cell responses by inhibiting IL-2 production. In this report, using an Ag-specific TCR transgenic system, we show that naive T cells stimulated with cognate Ag in the presence of preactivated CD4(+)CD25(+) T cells also become refractory to the mitogenic effects of IL-2. T cells stimulated in the presence of regulatory T cells up-regulated high affinity IL-2R, but failed to produce IL-2, express cyclins or c-Myc, or exit G(0)-G(1). Exogenous IL-2 failed to break the mitotic block, demonstrating that the IL-2 production failure was not wholly responsible for the proliferation defect. This IL-2 unresponsiveness did not require the continuous presence of CD4(+)CD25(+) regulatory T cells. The majority of responder T cells reisolated after coculture with regulatory cells failed to proliferate in response to IL-2, but were not anergic and proliferated in response to Ag. The mitotic block was also dissociated from the antiapoptotic effects of IL-2, because IL-2 still promoted the survival of T cells that had been cocultured with CD4(+)CD25(+) T cells. IL-2-induced STAT5 phosphorylation in the cocultured responder cells was intact, implying that the effects of the regulatory cells were downstream of receptor activation. Our results therefore show that T cell activation in the presence of CD4(+)CD25(+) regulatory T cells can induce an alternative stimulation program characterized by up-regulation of high affinity IL-2R, but a failure to produce IL-2, and uncoupling of the mitogenic and antiapoptotic effects of IL-2.     

CD31 is required on CD4+ T cells to promote T cell survival during Salmonella infection

Hematopoietic cells constitutively express CD31/PECAM1, a signaling adhesion receptor associated with controlling responses to inflammatory stimuli. Although expressed on CD4(+) T cells, its function on these cells is unclear. To address this, we have used a model of systemic Salmonella infection that induces high levels of T cell activation and depends on CD4(+) T cells for resolution. Infection of CD31-deficient (CD31KO) mice demonstrates that these mice fail to control infection effectively. During infection, CD31KO mice have diminished numbers of total CD4(+) T cells and IFN-γ-secreting Th1 cells. This is despite a higher proportion of CD31KO CD4(+) T cells exhibiting an activated phenotype and an undiminished capacity to prime normally and polarize to Th1. Reduced numbers of T cells reflected the increased propensity of naive and activated CD31KO T cells to undergo apoptosis postinfection compared with wild-type T cells. Using adoptive transfer experiments, we show that loss of CD31 on CD4(+) T cells alone is sufficient to account for the defective CD31KO T cell accumulation. These data are consistent with CD31 helping to control T cell activation, because in its absence, T cells have a greater propensity to become activated, resulting in increased susceptibility to become apoptotic. The impact of CD31 loss on T cell homeostasis becomes most pronounced during severe, inflammatory, and immunological stresses such as those caused by systemic Salmonella infection. This identifies a novel role for CD31 in regulating CD4 T cell homeostasis.     

Immunodominance analysis through interactions of CD8+ T cells and DCs in lymph nodes

Immunodominance is a common phenomenon observed in multiple epitopes immune systems. Previous studies hypothesize that the competition among CD8⁺ T cell responses against different epitopes can be used to explain immunodominance. This paper proposes a mathematical model that describes the dynamics of CD8⁺ T cells primed by antigen-presenting dendritic cells (DCs) in the lymph nodes, and shows that the overall avidity of the interactions between peptide-specific T cells and cognate antigen-bearing DCs may determine the immunodominance. The model suggests the probability that a peptide-specific T cell be immunodominant is proportional to (1) the cognate T cell receptor (TCR) affinity, (2) the number of complexes of cognate peptide and major histocompatibility complex (pMHC) per DC, and (3) the half-life of cognate peptide-specific pMHC. The model predicts a threshold density of pMHC complexes for T cell activation. These observations from the mathematical model are consistent with experimental studies in the open literature. For DC-based vaccine design, the model suggests a strategy of immunotherapy based on the injection of cognate antigen-pulsed DCs.     

Persistent high frequencies of varicella-zoster virus ORF4 protein-specific CD4+ T cells after primary infection

Open reading frame 4 (ORF4) of varicella-zoster virus (VZV) encodes an immediate-early protein that is believed to be important for viral infectivity and establishing latency. Evidence suggests that VZV-specific T cells are crucial in the control of viral replication, but there are no data addressing the existence of potential ORF4 protein-specific CD4+ T cells. We tested the hypothesis that VZV ORF4 protein-specific CD4+ T cells could be identified and characterized within the peripheral blood of healthy immune donors following primary infection. Gamma interferon (IFN-gamma) immunosorbent assays were used to screen peripheral blood mononuclear cells obtained from healthy seropositive donors for responses to overlapping ORF4 peptides, viral lysate, and live vaccine. High frequencies of ORF4 protein-specific T cells were detected ex vivo in individuals up to 52 years after primary infection. Several immunogenic regions of the ORF4 protein were identified, including a commonly recognized epitope which was restricted through HLA-DRB1*07. Total ORF4 protein-specific responses comprised 19.7% and 20.7% of the total lysate and vaccine responses, respectively, and were dominated by CD4+ T cells. Indeed, CD4+ T cells were found to dominate the overall virus-specific IFN-gamma cellular immune response both ex vivo and after expansion in vitro. In summary, we have identified an ORF4 protein as a novel target antigen for persistent VZV-specific CD4+ T cells, with implications for disease pathogenesis and future vaccine development.     

Contrasting effects of low-dose IL-2 on vaccine-boosted simian immunodeficiency virus (SIV)-specific CD4+ and CD8+ T cells in macaques chronically infected with SIVmac251

IL-2, the first cytokine discovered with T cell growth factor activity, is now known to have pleiotropic effects on T cells. For example, it can promote growth, survival, and differentiation of Ag-selected cells, or facilitate Ag-induced cell death of T cells when Ag persists, and in vivo, it is thought to contribute to the regulation of the size of adaptive T cell response. IL-2 is deficient in HIV-1 infection and has been used in the management of HIV-1-infected individuals undergoing antiretroviral therapy. In this study, we investigated how continuous low-dose IL-2 affected the CD4+ and CD8+ T cell response induced by two inoculations of a canarypox recombinant SIV-based vaccine candidate in healthy macaques chronically infected with SIVmac251. These macaques had normal levels of CD4+ T cells at the beginning of antiretroviral therapy treatment. Vaccination in the presence of IL-2 significantly augmented Gag-specific CD8+ T cell responses, but actually reduced Gag-specific CD4+ T cell responses. Although IL-2 at low doses did not change the overall concentration of circulating CD4+ or CD8+ T cells, it expanded the frequency of CD4+CD25+ T cells. Depletion of the CD4+CD25+ T cells in vitro, however, did not result in a reconstitution of Gag-specific CD4+ responses or augmentation of SIV-specific CD8+ T cell responses. Thus, we conclude that the decrease in virus-specific CD4+ T cell response may be due to IL-2-promoted redistribution of cells from the circulation, or due to Ag-induced cell death, rather than suppression by a T regulatory population.     

CD4+CD25+ regulatory T cells control the progression from periinsulitis to destructive insulitis in murine autoimmune diabetes

Non-obese diabetic (NOD) mice develop spontaneous T-cell responses against pancreatic beta-cells, leading to islet cell destruction and diabetes. Despite high genetic similarity, non-obese resistant (NOR) mice do not develop diabetes. We show here that spleen cells of both NOD and NOR mice respond to the islet cell antigen glutamic acid decarboxylase-65 in IFN-gamma-ELISPOT assays. Moreover, NOR-T cells induce periinsulitis in NOD SCID recipient mice. Thus, a potentially pathogenic islet cell-specific T-cell response arises in NOR and NOD mice alike; the mechanism that prevents the autoimmune progression of self-reactive T cells in NOR mice presumably acts at the level of effector function. Consistent with this hypothesis, CD4+CD25+ cell-depleted spleen cells from NOR mice mediated islet cell destruction and overt diabetes in NOD SCID mice. Therefore, islet cell-specific effector cells in NOR mice appear to be under the control of CD4+CD25+ regulatory T cells, confirming the importance of regulatory cells in the control of autoimmune diabetes.