Generation of peptide-specific CD8+ T cells by phytohemagglutinin-stimulated antigen-mRNA-transduced CD4+ T cells

Functional analysis of antigen-specific CD8(+) T cells is important for understanding the immune response in various immunological disorders. To analyze CD8(+) T cell responses to a variety of antigens with no readily defined peptides available, we developed a system using CD4(+) phytohemagglutinin (PHA) blasts transduced with mRNA for antigen molecules. CD4(+) PHA blasts express MHC class I and II, and also CD80 and CD86 and are thus expected to serve as potent antigen presenting cells. EGFP mRNA could be transduced into and the protein expressed by more than 90% of either LCL or CD4(+) PHA blasts. Its expression stably persisted for more than 2 weeks after transduction. In experiments with HLA-A*2402 restricted CD8(+) CTL clones for either EBNA3A or a cancer-testis antigen, SAGE, mRNA-transduced lymphoid cells were appropriate target cells in ELISPOT assays or (51)Cr releasing assays. Finally, using CD4(+) PHA blasts transduced with mRNA of a cancer-testis antigen MAGE-A4, we successfully generated specific CTL clones that recognized a novel HLA-B*4002 restricted epitope, MAGE-A4(223-231). Messenger RNA-transduced CD4(+) PHA blasts are thus useful antigen presenting cells for analysis of CD8(+) T cell responses and induction of specific T cells for potential immunotherapy.     

Evidence of productively infected CD8+ T cells in patients with AIDS: implications for HIV-1 pathogenesis

CD8+ T lymphocytes play an important protective role against HIV infection. The onset of AIDS is associated with a decline in both the number of CD8+ T lymphocytes and anti-HIV cytotoxic activity in CD8+ T cells. The reason for this progressive failure of CD8+ T cells in HIV-1 infection remains unknown. Earlier reports have shown presence of viral DNA in CD8+ cells of HIV-1-infected patients; under some conditions, CD8+ T cells have been shown to express CD4 in vitro and can be susceptible to infection with HIV-1. However, whether CD8+ lymphocytes in vivo can be productively infected with HIV-1 remains unclear. In this study, we generated multiple CD8+ T-cell clones from two patients with AIDS. These clones were CD8+/CD3+ but did not express CD4. Several of these CD8+ clones from both patients were found to be endogenously infected with HIV-1 and spontaneously produced these viruses. CD8+ cell-produced HIV-1 was biologically competent because viruses produced by most of these clones could efficiently infect and replicate in peripheral blood lymphocytes from HIV-negative donors. In addition, some of these viruses were able to form syncytia in MT-2 cells indicating syncytium-inducing phenotype. Comparison of the sequences in V3 loop areas among different viruses showed changes in some of the clones from both patients. For the first time, this report provides direct evidence that mature CD8+ T cells can be productively infected with HIV-1 in patients with AIDS. Direct infection of CD8+ T lymphocytes may play a role in the eventual failure of these cells in HIV-1 infection.     

CD4+T cells restricted by DQ not by DR support CD8+T cells to grow

Much attention has been paid whether there are any differences in regulating the human immune response between HLA-DR and -DQ molecules encoded by the genes within the HLA class II multigene family. Previous studies have suggested that HLA DQ molecules control low responsiveness through activating CD4 T cells which generate CD8 positive T cells, whereas HLA -DR molecules control high responsiveness through activating CD4 helper T cells. To examine this model we investigated the streptococcal cell wall antigen (SCW) specific T cell lines restricted by either DR or DQ molecule. To identify the restricting molecules, L cell transfectants expressing DQw1, DR2AB1 or DR2AB5 from Dw12 haplotype or DQw4, DR4 or DRw53 from DW15 haplotype were used. 1. From individuals with Dw12 which is a low responder haplotype to SCW, T cell clones specific to SCW and restricted by HLA-DQw1 or DR2 were identified, whereas from individuals with Dw15 which is a high responder haplotype, only DR4 or DRw53 restricted T cell clones were identified and DQw4 restricted T cells were never observed. 2. SCW specific CD4 T cells restricted by DQw1 were able to support the growth of CD8 positive cells, whereas those restricted by DR4 could not do so. 3. The CD8 T cells also required autologous antigen presenting cells and SCW to grow, and they completely blocked the immune response to SCW in vitro. These observations clearly demonstrated the distinct function of HLA-DQ and -DR molecules in regulating the human immune response to SCW.     

A vaccinia-based elispot assay for detection of CD8+ T cells from HIV-1 infected children

HIV-specific CD8+ T lymphocytes participate in the control of viral replication in infected patients. These responses are of low intensity in young infants and are decreased by antiretroviral therapy. In the present study, we report on a recombinant Vaccinia virus (rVV)-based Elispot assay for the detection of HIV-specific CD8+ T cells immediately after isolation of peripheral blood mononuclear cells (PBMC). The rVV-based assay was highly sensitive; 48 out of 50 children had a positive response against the rVV encoding HIV Env-Gag-Pol antigen. Interferon-gamma was produced by CD8+ T cells, and CD14+/15+ cells were the main cell subset presenting antigens expressed by rVV. We observed that the cell input per well had a critical influence on the sensitivity of the assay. Results from the ex vivo Elispot assay correlated poorly with those of the 51Cr release assay performed after expansion of PBMC in vitro; thus, both assays gave information on different subsets and/or functions of the HIV-specific T cell response.     

Immunodominant HIV-1-specific HLA-B- and HLA-C-restricted CD8+ T cells do not differ in polyfunctionality

HIV-1 specific HLA-B-restricted CD8+ T cell responses differ from HLA-C-restricted responses in antiviral effectiveness. To investigate possible reasons for these differences, we characterized the frequency and polyfunctionality of immmunodominant HLA-B*57/B5801- and HLA-Cw*07-restricted CD8+ T cells occurring concurrently in nine study subjects assessing IFN-γ, TNF-α, IL-2, MIP-1β, and CD107a by flow cytometry and analyzed sequence variation in targeted epitopes. HLA-B*57/5801 and HLA-Cw*07 restricted CD8+ T cells did not differ significantly in polyfunctionality (p = 0.84). Possession of three or more functions correlated positively with CD4+ T cell counts (r = 0.85; p = 0.006) and monofunctional CD8+ T cells inversely correlated with CD4 cell counts (r = −0.79; p = 0.05). There were no differences in polyfunctionality of CD8+ T cells specific to wildtype versus mutated epitopes. These results suggest that loss of polyfunctionality and increase in monofunctional HIV-1-specific CD8+ T cells are associated with disease progression independent of restricting HLA allele. Furthermore, sequence variation does not appear to significantly impact CD8+ T cell polyfunctionality in chronic HIV-1 infection.     

Evaluation of HIV-1 p24 antigenemia and level of CD8+CD38+ T cells as surrogate markers of HIV-1 RNA viral load in HIV-1-infected patients in Dakar, Senegal

Alternative, affordable, and simple assays to monitor antiretroviral therapy (ART) in resource-poor settings are needed. We have evaluated and compared a heat-denatured (HD) HIV p24 amplified enzyme-linked immunosorbent assay from Perkin-Elmer and CD38CD8 T-cell levels, determined by flow cytometry, for their capacity to predict viral load (VL) in HIV-1-infected patients from Senegal. Median fluorescence intensity (MFI) of CD38 expression on memory (CD45RO) CD8 T cells correlated better with RNA VL than HD p24 antigenemia (R = 0.576, P < 0.0001 vs R = 0.548, P < 0.0001). MFI of CD38 expression on memory CD8 T cells could predict detectable RNA VL (VL = 2.6 log10) with a sensitivity of 87% and a specificity of 74%. A comparable sensitivity (89%) could be reached for HD p24 assay, but only to predict RNA VL of more than 5 logs, which might lead to unacceptable delays in clinical decision making. The clinical use of the HD p24 assay to monitor ART in Senegal would require more comparative data about the kinetics of p24 antigen and HIV RNA in peripheral blood as well as further evaluation regarding its sensitivity toward subtype A and CRF02. MFI of CD38 expression on memory CD8 T cells appeared to be a better alternative to monitor ART in HIV-infected patients from Senegal.     

TGF-beta1 production by CD4+ CD25+ regulatory T cells is not essential for suppression of intestinal inflammation

Naturally occurring CD4+ CD25+ regulatory T cells (Treg) are potent suppressors of CD4+ and CD8+ T cell responses in vitro and inhibit several organ-specific autoimmune diseases. While most in vitro studies suggest that CD4+ CD25+ Treg cells adopt a cytokine-independent but cell contact-dependent mode of T cell regulation, their precise mechanism of suppression in vivo remains largely unknown. Here we examine the functional contribution of Treg cell-derived TGF-beta1 and effector T cell responsiveness to TGF-beta in CD4+ CD25+ T cell-mediated suppression of inflammatory bowel disease (IBD). We show that CD4+ CD25+ Treg cells from either TGF-beta1+/+ or neonatal TGF-beta1-/- mice can suppress the incidence and severity of IBD as well as colonic IFN-gamma mRNA expression induced by WT CD4+ CD25- effector T cells. Furthermore, TGF-beta-resistant Smad3-/- CD4+ CD25+ Treg cells are equivalent to WT Treg cells in their capacity to suppress disease induced by either WT or Smad3-/- CD4+ CD25- effector T cells. Finally, anti-TGF-beta treatment exacerbates the colitogenic potential of CD4+ CD25- effector T cells in the absence of CD4+ CD25+ Treg cells. Together, these data demonstrate that in certain situations CD4+ CD25+ T cells are able to suppress intestinal inflammation by a mechanism not requiring Treg cell-derived TGF-beta1 or effector T cell/Treg cell responsiveness to TGF-beta via Smad3.     

Increased IFN-gamma production by NK and CD3+/CD56+ cells in sexually HIV-1-exposed but uninfected individuals

The mechanisms involved in controlling the establishment of HIV-1 infection are not fully understood. In particular, the role of innate immunity in natural resistance exhibited by individuals who are continuously exposed to HIV-1 but remain seronegative (ESN) has not been thoroughly evaluated. We determined the frequency and function of peripheral blood innate immune cells (plasmacytoid and myeloid dendritic cells, monocytes, NK cells, CD3+/CD56+ cells and invariant NKT cells) in ESN, chronically HIV-1-infected and low-risk HIV-1 seronegative individuals. ESN demonstrated a similar frequency of innate immune cells in comparison to controls and a higher frequency of dendritic cells, NK and invariant NKT cells compared to HIV-1-infected subjects. Incubation of mononuclear cells with stimulatory CpG ODN induced CD86 and CD69 up-regulation to a similar degree on innate cells from the three study groups. CpG ODN-stimulated secretion of cytokines was also similar between ESN and controls, while secretion of IFN-alpha was significantly decreased in HIV-1+ individuals. Importantly, expression of IFN-gamma by PMA/Ionomycin-activated CD56(bright) NK cells and CD3+/CD56+ cells was significantly higher in ESN when compared with controls. The anti-viral effects of IFN-gamma are well established, and so our results suggest that IFN-gamma production by innate immune cells might be one of the multiple factors involved in controlling the establishment of sexually transmitted HIV-1 infection.     

'Ignorance' of antigen-specific murine CD4+ and CD8+ T cells is overruled by lipopolysaccharide and leads to specific induction of IFN-gamma

Lipopolysaccharide (LPS) can activate human and murine T cells in vivo and in vitro. Here we analysed the effects of LPS on T cells with defined specificities in T-cell receptor (TCR)-transgenic systems. LPS rapidly induced high amounts of interferon (IFN)-gamma in a subpopulation of purified T cells from DO11.10 (OVA323-339/H2-Ad) and OT-1 (OVA257-264/H2-Kb) mice when coincubated with antigen-pulsed peritoneal exudate cells (PECs). LPS induced IFN-gamma in T cell cultures even when the number of antigenic major histocompatibility complex (MHC) class-I complexes was too small to stimulate the T cells. LPS, thus, overruled the unresponsiveness of the otherwise 'antigen-ignorant' T cells. The release of IFN-gamma strictly correlates with the PECs' ability to produce interleukin (IL)-12. In contrast to the induction of IFN-gamma, antigen-specific IL-2 secretion and proliferation of T cells were rather decreased in the presence of LPS. Only very few IFN-gamma-secreting natural killer (NK) cells and natural killer T (NKT) cells in the given experimental system could be detected using intracellular fluorescence-activated cell sorter (FACS) staining. Taken together, our results indicate that LPS has the potential to activate quiescent T cells and to specifically induce IFN-gamma in CD4 and CD8 T cells. This may have direct consequences for the activation of autoreactive T cells following bacterial infections.     

CD20+ T cell numbers are decreased in untreated HIV-1 patients and recover after HAART

To elucidate if CD20(+) T cells are affected by HIV-1 infection and may have a prognostic value for the course of disease, numbers of CD20(+) T cells were determined in healthy controls, untreated and HAART-treated HIV-1 patients. Coexpression patterns of CD4, CD8, and CD38 were analysed on CD3(+)CD20(+) and CD3(+)CD20(-) T cells. We found a significant decrease of CD20(+) T cell numbers in untreated HIV-1 patients (1.4%) as compared to healthy controls (2.5%) which recovered under HAART (1.9%). Particularly, the CD8(+) T cell compartment was affected revealing significant differences between healthy controls (3.4%) and both treated (1.7%) and untreated (1.1%) patients. CD38 was expressed on a few CD20(+) T cells but preferentially on CD20(-) cells in all three groups. IFN-γ production was measured upon cell activation using PMA alone or in combination with ionomycin in order to assess functional capacities of the cells. PMA alone was much more effective in CD20(+) cells regardless of CD38 coexpression, indicating a supportive role of CD20 but not CD38 in T cell activation. Here we present data showing that CD3(+)CD20(+) T cells are decreased in untreated HIV-1 patients and normal numbers are restored under HAART. Expression of CD20 and CD38 is independently regulated on T cells. Contrary to CD38, CD20 can substitute ionophores for Ca(2+) flux in early T cell activation and also strongly amplify cell stimulation in the presence of Ca(2+) ionophores, indicating that CD20 contributes to T cell activation.     

CD8+ cytolytic T lymphocytes become infected in vitro in the process of killing HIV-1-infected target cells

In the present study the requirements for in vitro infection of antigen-specific CD8⁺ cytotoxic T lymphocytes (CTL) with human immunodeficiency virus –1(HIV-1) were investigated. CD3⁺CD8⁺CD4^? HIV-1 nef-specific CTL become infected with HIV-1 after short-term co-culture with HLA-matched HIV-1-infected CD20⁺ B lymphoblastoid cells (B-LCL) which are specifically killed. Similar results were observed with an allospecific CD8⁺ CTL population. In addition, co-culture experiments showed that once infected with HIV-1, these CD8⁺ CTL could spread the infection further to uninfected CD4⁺ lymphocytes. In contrast, CD8⁺ CTL did not become infected with HIV-1 when co-cultured with HLA-mismatched HIV-1-infected B-LCL which are not killed. These observations in vitro could have relevance in peripheral lymphoid organs contributing to the progressive decrease of HIV-specific CD8⁺ CTL activity that is associated with the progression to AIDS.     

Enumeration of latently infected CD4+ T cells from HIV-1-infected patients using an HIV-1 antigen ELISPOT assay.

BACKGROUND: Latently infected resting CD4(+) T cells carrying replication-competent HIV-1 are present in naive, chronically infected individuals as well as in those who are receiving highly active antiretroviral therapy (HAART). These cells serve as a potential source of reactivation of viral replication and remain a major obstacle for the eradication of HIV-1. OBJECTIVES: The enzyme-linked immunospot (ELISPOT) assay was adapted to the detection and the enumeration of HIV-1 antigen-secreting cells at the single cell level. We applied this test to count latently HIV-1-infected CD4(+) T cells. STUDY DESIGN: Latently infected CD4(+) T cells were assessed in an in vitro model of HIV-1-infected resting CD4(+) T cells as well as in eighteen HAART-treated and in four HIV-1-infected untreated patients. Enriched CD4(+) T cells were cultured with or without antibodies against CD3 and CD28 T cell receptors and with irradiated peripheral blood mononuclear cells from HIV-1 seronegative individuals. At the term of the cell culture, CD4(+) T lymphocytes were tested using the HIV-1 antigen ELISPOT assay. RESULTS: In the experimental HIV-1 infection model, 5579+/-4190 CD4(+) T cells secreting HIV-1 antigen were enumerated after polyclonal activation. In contrast, only 15+/-6 HIV-1 immunospots were obtained from unstimulated T cells. In all patients tested, induced HIV-1 antigen-secreting cells were measured at a frequency of 55+/-108/10(6) CD4(+) T cells. CONCLUSION: As each immunospot represents one HIV-1 antigen-secreting cell, the HIV-1 ELISPOT assay is a powerful to enumerate circulating CD4(+) T lymphocytes latently infected with HIV-1.     

Virtual memory CD8+ T cells restrain the viral reservoir in HIV-1-infected patients with antiretroviral therapy through derepressing KIR-mediated inhibition

The viral reservoir is the major hurdle in developing and establishing an HIV cure. Understanding factors affecting the size and decay of this reservoir is crucial for the development of therapeutic strategies. Recent work highlighted that CD8+ T cells are involved in the control of viral replication in ART-treated HIV-1-infected individuals, but how CD8+ T cells sense and restrict the HIV reservoir are not fully understood. Here, we demonstrate that a population of unconventional CD45RA+, PanKIR+, and/or NKG2A+ virtual memory CD8+ T cells (T_VM cells), which confer rapid and robust protective immunity against pathogens, plays an important role in restraining the HIV DNA reservoir in HIV-1-infected patients with effective ART. In patients undergoing ART, T_VM cells negatively correlate with HIV DNA and positively correlate with circulating IFN-α2 and IL-15. Moreover, T_VM cells constitutively express high levels of cytotoxic granule components, including granzyme B, perforin and granulysin, and demonstrate the capability to control HIV replication through both cytolytic and noncytolytic mechanisms. Furthermore, by using an ex vivo system, we showed that HIV reactivation is effectively suppressed by T_VM cells through KIR-mediated recognition. This study suggests that T_VM cells are a promising target to predict posttreatment virological control and to design immune-based interventions to reduce the reservoir size in ART-treated HIV-1-infected individuals.     

Cross-priming of CD8+ T cells by viral and tumor antigens is a robust phenomenon

"Cross-priming" refers to the activation of naive CD8+ T cells by antigen-presenting cells that have acquired nominal antigens from another cell. The biological relevance of cross-priming of CD8+ T cells has recently been challenged (Zinkernagel, R. M., Eur. J. Immunol. 2002. 32: 2385-2392), on the basis that responses are weak or poorly quantitated, and the determinants recognized are undefined. Here we show that cross-priming is a robust process that elicits vigorous primary responses to multiple peptides in two well-defined systems. Our findings support the relevance of cross-priming in CD8+ T cell responses to viruses and tumor cells, and demonstrate that cross-priming elicits CD8+ T cells to determinants generated by the endogenous processing pathway.     

CD8+ regulatory T cells in persistent human viral infections

Regulatory T cells (T(reg) cells) play an important role in the regulation and suppression of immune responses to self- and foreign antigens. Suppressed and impaired host immune responses are a major characteristic of many persistent human virus infections, such as those caused by human immunodeficiency virus (HIV), hepatitis C virus (HCV), and herpes virus. It has recently become evident that immune regulation mediated by T(reg) cells may comprise one mechanism that contributes to the impairment of virus-specific immune responses. Indeed, during viral infection, the generation of distinct subsets of CD4+ as well as CD8+ T(reg) cells has been reported. The phenotypic and functional heterogeneity of T(reg) cell subsets involved in the suppression of virus-specific immune responses suggests that different mechanisms and factors contribute to the generation of those cells during viral infection. This review focuses on the CD8+ T(reg) cell subset and summarizes current knowledge about the induction and function of CD8+ T(reg) cells in persistent human virus infections.     

Notch signalling regulates cytokine production by CD8+ and CD4+ T cells

The Notch signalling pathway regulates several aspects of cellular differentiation such as T lineage commitment and effector functions on peripheral T cells; however, there is limited information regarding Notch receptor expression on different T cell subsets and the putative role of the different receptors on T cell effector function. Here, we studied the protein expression of Notch receptors on murine T cells in vitro and in vivo and analysed the role of the Notch pathway in cytokine production by CD4+ and CD8+ T cells. We found that resting CD4+ and CD8+ T cells do not express Notch receptors, but they upregulate Notch 1 and Notch 2 shortly after in vitro and in vivo activation. Using a γ-secretase inhibitor, which blocks Notch signalling through all Notch receptors, we demonstrated that the Notch pathway regulates IL-10 production by CD4+ T cells and IFN-γ and IL-17 production by CD8+ T cells. These results suggest that Notch 1 and 2 are expressed by CD4+ and CD8+ T cells and represent the putative Notch receptors that regulate effector functions and cytokine production by these cells.     

Antigen-specific CD8+ T cells inhibit IgE responses and interleukin-4 production by CD4+ T cells

There is a growing body of evidence which suggests that CD8⁺ T cells play an important part in regulating the IgE response to non-replicating antigens. In this study we have systematically investigated their role in the regulation of IgE and of CD4⁺ T cell responses to ovalbumin (OVA) by CD8⁺ T cell depletion in vivo. Following intraperitoneal immunization with alum-precipitated OVA, OVA-specific T cell responses were detected in the spleen and depletion of CD8⁺ T cells in vitro significantly enhanced the proliferative response to OVA. Depletion of CD8⁺ T cells in vivo 7 days after immunization failed to enhance IgE production, while depletion of CD8⁺ T cells on days 12–18 greatly enhanced the IgE response, which rose to 26 μ/ml following a second injection of anti-CD8 on day 35 and remained in excess of 1 μ/ml over 300 days afterwards. Reconstitution on day 21 of rats CD8-depleted on day 12 with purified CD8⁺ T cells from animals immunized on day 12 completely inhib ited the IgE response. This effect was antigen specific; CD8⁺ T cells from OVA-primed animals had little effect on the IgE response of bovine serum albumin immunized rats. In vivo, CD8⁺ T cell depletion decreased interferon (IFN)-γ production but enhanced interleukin (IL)-4 production by OVA-stimulated splenic CD4⁺ T cells. Furthermore, CD8⁺ T cell depletion and addition of anti-IFN-γ antibody enhanced IgE production in vitro in an IL-4-supplemented mixed lymphocyte reaction. These data clearly show that antigen-specific CD8⁺ T cells inhibit IgE in the immune response to non-replicating antigens. The data indicate two possible mechanisms: first, CD8⁺ T cells have direct inhibitory effects on switching to IgE in B cells and second, they inhibit OVA-specific IL-4 production but enhance IFN-γ production by CD4⁺ T cells.