Synaptic transfer by human gamma delta T cells stimulated with soluble or cellular antigens

B, alpha beta T, and NK lymphocytes establish immunological synapses (IS) with their targets to enable recognition. Transfer of target cell-derived Ags together with proximal molecules onto the effector cell appears also to occur through synapses. Little is known about the molecular basis of this transfer, but it is assumed to result from Ag receptor internalization. Because human gamma delta T cells recognize soluble nonpeptidic phosphoantigens as well as tumor cells such as Daudi, it is unknown whether they establish IS with, and extract molecules from, target cells. Using flow cytometry and confocal microscopy, we show in this work that Ag-stimulated human V gamma 9/V delta 2 T cells conjugate to, and perform molecular transfer from, various tumor cell targets. The molecular transfer appears to be linked to IS establishment, evolves in a dose-dependent manner in the presence of either soluble or cellular Ag, and requires gamma delta TCR ligation, Src family kinase signaling, and participation of the actin cytoskeleton. Although CD45 exclusion characterized the IS performed by gamma delta T cells, no obvious capping of the gamma delta TCR was detected. The synaptic transfer mediated by gamma delta T cells involved target molecules unrelated to the cognate Ag and occurred independently of MHC class I expression by target cells. From these observations, we conclude that despite the particular features of gamma delta T cell activation, both synapse formation and molecular transfer of determinants belonging to target cell characterize gamma delta T cell recognition of Ags.     

Optimization of methods for peripheral blood mononuclear cells isolation and expansion of human gamma delta T cells

Human Vg9/Vδ2 T cells (γδ T cells) are immune surveillance cells both in innate and adaptive immunity and are a possible target for anticancer therapies, which can induce immune responses in a variety of cancers. Small non-peptide antigens such as zoledronate can do activation and expansion of T cells in vitro. It is evident that for adoptive cancer therapies, large numbers of functional cells are needed into cancer patients. Hence, optimization of methods needs to be carried out for the efficient expansion of these T cells. Standardization of peripheral blood mononuclear cells (PBMCs) isolation was devised. Cytokines (interleukin 2 (IL-2) and interleukin 15 (IL-15)) and zoledronate were also standardized for different concentrations. It was found that an increased number of PBMCs were recovered when washing was done at 1100 revolution per minute (rpm). Significantly high expansion fold was (2524 ± 787 expansion fold) achieved when stimulation of PBMCs was done with 1 µM of zoledronate and both cytokines IL-2 and IL-15 supported the expansion and survival of cells at the concentrations of 100 IU/ml and 10 ng/ml respectively. 14-day cultures showed highly pure (91.6 ± 5.1%) and live (96.5 ± 2.5%) expanded γδ T cells. This study aimed to standardize an easy to manipulate technique for the expansion of γδ T cells, giving a higher yield.     

Extensive replicative capacity of human central memory T cells

To characterize the replicative capacity of human central memory (T(CM)) CD4 T cells, we have developed a defined culture system optimized for the ex vivo expansion of Ag-specific CD4(+) T cells. Artificial APCs (aAPCs) consisting of magnetic beads coated with Abs to HLA class II and a costimulatory Ab to CD28 were prepared; peptide-charged HLA class II tetramers were then loaded on the beads to provide Ag specificity. Influenza-specific DR*0401 CD4 T(CM) were isolated from the peripheral blood of normal donors by flow cytometry. Peptide-loaded aAPC were not sufficient to induce resting CD4 T(CM) to proliferate. In contrast, we found that the beads efficiently promoted the growth of previously activated CD4 T(CM) cells, yielding cultures with >80% Ag-specific CD4 cells after two stimulations. Further stimulation with peptide-loaded aAPC increased purity to >99% Ag-specific T cells. After in vitro culture for 3-12 wk, the flu-specific CD4 T(CM) had surface markers that were generally consistent with an effector phenotype described for CD8 T cells, except for the maintenance of CD28 expression. The T(CM) were capable of 20-40 mean population doublings in vitro, and the expanded cells produced IFN-gamma, IL-2, and TNF-alpha in response to Ag, and a subset of cells also secreted IL-4 with PMA/ionomycin treatment. In conclusion, aAPCs expand T(CM) that have extensive replicative capacity, and have potential applications in adoptive immunotherapy as well as for studying the biology of human MHC class II-restricted T cells.     

Structural features of nonpeptide prenyl pyrophosphates that determine their antigenicity for human gamma delta T cells.

Human Vgamma2Vdelta2(+) T cells proliferate in vivo during many microbial infections. We have found that Vgamma2Vdelta2(+) T cells recognize nonpeptide prenyl pyrophosphates and alkylamines. We now have defined structural features that determine the antigenicity of prenyl pyrophosphates by testing synthetic analogs for bioactivity. We find that the carbon chain closest to the pyrophosphate moiety plays the major role in determining bioactivity. Changes in this area, such as the loss of a double bond, abrogated bioactivity. The loss of a phosphate from the pyrophosphate moiety also decreased antigenicity 100- to 200-fold. However, nucleotide monophosphates could be added with minimal changes in bioactivity. Longer prenyl pyrophosphates also retained bioactivity. Despite differences in CDR3 sequence, Vgamma2Vdelta2(+) clones and a transfectant responded similarly. Ag docking into a Vgamma2Vdelta2 TCR model reveals a potential binding site in germline regions of the Vgamma2Jgamma1.2 CDR3 and Vdelta2 CDR2 loops. Thus, Vgamma2Vdelta2(+) T cells recognize a core carbon chain and pyrophosphate moiety. This recognition is relatively unaffected by additions at distal positions to the core Ag unit.     

Gamma delta T cells from HIV+ donors can be expanded in vitro by zoledronate/interleukin-2 to become cytotoxic effectors for antibody-dependent cellular cytotoxicity

Background aimsImmunotherapy using γδ T cells capable of mediating antibody-dependent cellular cytotoxicity (ADCC) is a promising anti-human immunodeficiency virus (HIV) strategy. Approved aminobispohsphonate drugs, for example zoledronate (Zometa), stimulate γδ T cells in cancer patients, where they may promote direct tumor killing. Knowing that γδ T cells are modulated during HIV disease, documenting their responses and potential for controlling HIV is important. We investigated whether zoledronate/interleukin (IL)-2 could expand cytotoxic Vδ2 cells from HIV⁺ donors and whether these cells functioned in ADCC.MethodsPeripheral blood mononuclear cells from uninfected controls and HIV⁺ individuals receiving anti-retroviral therapy were treated with isopentenyl pyrophosphate (IPP) or zoledronate plus IL-2 to expand the Vδ2⁺ subset. Immunophenotyping and functional analyzes (cytotoxicity or cytokine expression) allowed us to compare cell properties from individual donors and to compare the responses to each stimulating agent.ResultsZoledronate stimulated a greater expansion of Vδ2 cells in HIV⁺ individuals compared with phosphoantigen IPP, and these cells expressed CD16. CD56 expression (a marker for cytotoxic cells) was lower on zoledronate-expanded cells, consistent with significantly lower cytotoxicity against the Daudi tumor cell line. Cells expanded with either zoledronate or IPP were active in ADCC, were similar in terms of interferon (IFN)-γ and tumor necrosis factor (TNF)-α expression, and degranulated in response to Fc receptor cross-linking.ConclusionsZoledronate causes ex vivo expansion of Vδ2 cells from HIV⁺ individuals. Despite lower expression of CD56 and decreased direct cytotoxicity, these effectors were potent in ADCC. Zoledronate/IL-2- expanded cells have potential for immunotherapy to activate Vδ2 cells in HIV patients and enhance ADCC.     

Brief antigenic stimulation generates effector CD8 T cells with low cytotoxic activity and high IL-2 production

It is currently believed that a brief antigenic stimulation is sufficient to induce CD8 T cells to complete their differentiation program, become effector T cells, and subsequently generate memory. Because this concept was derived from studies in which only a single effector function was analyzed (either IFN-gamma production or target cell lysis), we wondered whether monitoring for multiple effector functions might reveal novel characteristics of effector CD8 T cells elicited by brief or prolonged Ag exposure. Using an in vitro system to generate effector T cells and an in vivo adoptive transfer model to track donor CD8 T cells, we found that the differentiation programs acquired by CD8 T cells after brief or prolonged antigenic stimulation were different. Although the frequencies of IFN-gamma and TNF-alpha producers were comparable for both effector CD8 T cell populations, there were major differences in cytotoxic potential and IL-2 production. Whereas prolonged (>24 h) Ag exposure stimulated effector CD8 T cells with high cytotoxic activity and low IL-2 production, brief (<24 h) stimulation generated effector CD8 T cells with low cytotoxic activity and high IL-2 production. The latter effector T cells rapidly converted into central memory-like CD8 T cells, exhibited long-term survival in adoptively transferred hosts, and gave robust recall responses upon Ag challenge. These data suggest that not all functions of effector CD8 T cells are equally inherited after brief or prolonged antigenic stimulation.     

Low-avidity self-specific T cells display a pronounced expansion defect that can be overcome by altered peptide ligands

Thymic expression of self-Ags results in the deletion of high-avidity self-specific T cells, but, at least for certain Ags, a residual population of self-specific T cells with low-affinity TCRs remains after negative selection. Such self-specific T cells are thought to play a role in the induction of T cell-mediated autoimmunity, but may also be used for the induction of antitumor immunity against self-Ags. In this study, we examine the functional competence of a polyclonal population of self-specific CD8+ T cells. We show that low-affinity interactions between TCR and peptide are associated with selective loss of critical T cell functions. Triggering of low levels of IFN-gamma production and cytolytic activity through low-affinity TCRs readily occurs provided high Ag doses are used, but IL-2 production and clonal expansion are severely reduced at all Ag doses. Remarkably, a single peptide variant can form an improved ligand for the highly diverse population of low-avidity self-specific T cells and can improve their proliferative capacity. These data provide insight into the inherent limitations of self-specific T cell responses through low-avidity TCR signals and the effect of modified peptide ligands on self-specific T cell immunity.     

MHC-unrestricted cytotoxic and proliferative responses of two distinct human gamma/delta T cell subsets to Daudi cells.

Human V gamma 9/V delta 2 T cells, the major subset of gamma/delta T cells in peripheral blood of adults, mediate proliferative and cytotoxic responses to Daudi Burkitt's lymphoma cells without previous in vitro exposure to Daudi. Our experiments show that some gamma/delta T cells coexpressing V gamma 9 and V delta 1 genes also react to Daudi cells in cytotoxic and proliferative assays. Expression of V gamma 9 is not sufficient for the recognition of Daudi cells because most gamma/delta T cells expressing V delta 1 paired with V gamma 9 or other V gamma genes neither kill Daudi cells nor proliferate to Daudi. V gamma 9/V delta 2 T cells do not proliferate to other cell lines such as K562 or Molt4 that are sensitive to MHC-unrestricted cytolysis by NK cells and by most IL-2-activated gamma/delta T cell clones. Cold target inhibition assays demonstrate that Daudi cells are stronger inhibitors than K562 and Molt4 of MHC-unrestricted lysis by V gamma 9/V delta 2 clones. However, cold Daudi cells are relatively weaker inhibitors of MHC-unrestricted lysis by NK cell clones, most gamma/delta T cell clones expressing V delta 1 and alpha/beta T cell clones. Thus, recognition by V gamma 9/V delta 2 T cells and certain V gamma 9/V delta 1 T cells of Daudi appears to involve a specific triggering pathway that is distinct from recognition by these gamma/delta T cells of Molt4, K562, and other target cells. NK cell clones and most other gamma/delta and alpha/beta T cell clones derived from the same normal volunteer blood donors do not show this specific interaction with Daudi cells. These data show that distinct subsets of human gamma/delta T cells recognize Daudi cells and support the idea that the gamma/delta TCR may be directly involved.     

Microanatomic clonality of gamma delta T cells in human leishmaniasis lesions.

T cells bearing gamma delta Ag receptors accumulate in the lesions of patients with localized American cutaneous leishmaniasis (LCL), and are thought to be involved in immunity to the parasite. To obtain clues as to the nature of the Ag recognized by these cells, we analyzed the diversity of the TCR delta-chain in LCL lesions. Using mAb against variable (V) encoded determinants with immunoperoxidase, both V delta 1 and V delta 2 subpopulations were identified in the dermal granulomas. However, within the epidermis of LCL lesions, the majority of the gamma delta T cells were V delta 1 positive. PCR analysis of lesion-derived DNA using oligonucleotide primers for V and junctional (J) gene segments revealed preferential usage of J delta 1 in lesions compared with the peripheral blood of these patients. Nucleotide sequence analysis of the V-J junction indicated limited diversity of gamma delta T cells within specific microanatomic regions. In addition, use of a single diversity (D) gene segment, D delta 3, in V delta 2 cells in lesions was observed, as opposed to multiple D delta gene segment usage in the blood of the same individuals. The distribution, gene segment usage and clonality of gamma delta T cells in lesions of leishmaniasis was remarkably similar to that observed in leprosy. Therefore, gamma delta T cells responding to infection may recognize a limited set of nominal Ag, perhaps common to distinct pathogens and/or those expressed by the host. Our findings are most consistent with a model in which specific gamma delta T cells are clonally selected by these Ag in lesions and undergo oligoclonal expansion within a microanatomic region.     

Essential requirement of antigen presentation by monocyte lineage cells for the activation of primary human gamma delta T cells by aminobisphosphonate antigen

Human gammadelta T cells respond to nonpeptide Ags such as pyrophosphomonoesters and alkylamines in a gammadelta TCR-dependent manner in the absence of other APCS: Recently, aminobisphosphonates such as pamidronate have also been shown to activate human gammadelta T cells. In the present study, we indicate that activation of primary gammadelta T cells by pamidronate strictly depends on the presence of monocyte-lineage cells, unlike that by pyrophosphomonoesters. Thus, although pamidronate induced cell clustering, proliferation, and IFN-gamma production of gammadelta T cells in the culture of PBMC, it failed to induce any of these activities in the culture of purified primary gammadelta T cells. By adding back the purified monocytes, however, both cell clustering and IFN-gamma production of gammadelta T cells by pamidronate could be restored. The pamidronate-pulsed, but not untreated, myelomonocytic line, THP-1, was capable of activating the purified gammadelta T cells to produce IFN-gamma, which was associated with the down-regulation of gammadelta TCR. Furthermore, pamidronate-pulsed THP-1 cells were significantly more susceptible to gammadelta T cell-mediated cytotoxicity than untreated THP-1. Also, TCR-defective Jurkat T cells transfected with gammadelta TCR genes produced a significant level of IL-2 in response to the pamidronate-pulsed THP-1 cells. These results have suggested strongly that human gammadelta T cells are functionally activated via gammadelta TCR by aminobisphosphonate Ag presented on the surface of monocyte lineage cells rather than directly by its free form.     

Leptospira interrogans activation of human peripheral blood mononuclear cells: preferential expansion of TCR gamma delta+ T cells vs TCR alpha beta+ T cells

Innate and adaptive immune responses induced by leptospirosis have not been well characterized. In this study we show that in vitro exposure of naive human PBMC to Leptospira interrogans results in cell proliferation and the production of IFN-gamma, IL-12, and TNF-alpha. Cell proliferation was highest when using high numbers of Leptospira. Optimal cell proliferation occurred at 6-8 days, and the majority of cells contained in these cultures were gamma/delta T cells. These cultures showed a 10- to 50-fold expansion of gamma/delta T cells compared with the initial cellular input. Additionally, these cultures contained elevated numbers of NK cells. In contrast, exposure of PBMC to low numbers of Leptospira failed to induce gammadelta T cell or NK cell expansion, but induced significant alphabeta T cell expansion. Vgamma9/Vdelta2 were expressed on all gamma/delta T cells expanded by exposure of PBMC to Leptorspira: Leptospira stimulation of purified TCRgammadelta(+) T cells, obtained from 8-day cultures of Leptospira-stimulated PBMC, induced high levels of IFN-gamma production, but no cell proliferation, suggesting that such stimulation of gammadelta T cells did not depend on specialized accessory cells or Ag processing. Finally, in patients with acute leptospirosis, there was a significant (4- to 5-fold) increase in the number of peripheral blood TCRgammadelta(+) T cells. These results indicate that Leptospira can activate gammadelta T cells and alphabeta T cells and will guide further investigations into the roles of these T cell populations in host defense and/or the pathology of leptospirosis.     

Ex vivo expansion of non-MHC-restricted cytotoxic effector cells as adoptive immunotherapy for myeloma

BACKGROUND: PBMC can be expanded ex vivo into aggressive cytotoxic effector cells (CEC) comprising T, NK and NKT cells. We identified the phenotype, cytotoxicity and mechanisms of killing of these CEC. METHODS: CY- and G-CSF-mobilized PBMC from myeloma patients were placed in Aim-V serum-free medium, IL-2 (50 IU/mL) and OKT-3 (50 ng/mL). Cytotoxicity was evaluated by selectively blocking the TCR, MHC class I or NKG2D receptor. RESULTS: The CEC expanded three-fold by day 7 and aggressively lysed myeloma cells (41.9%) compared with day 0 (4%; P=0.012). CD8+ CD56+ NKT cells performed the majority of lysis. The CD8+ cells greatly increased NKG2D expression during culture (P=0.005). Cytotoxicity correlated with target NKG2D ligand expression (P=0.0002). Blocking the TCR or MHC class I did not affect cytotoxicity (P>0.22). CD8+ cell-mediated lysis dropped 48% when the NKG2D receptor was blocked. Day 7 CEC aggressively lysed myeloma cells in an MHC- and non-MHC-restricted fashion, through the NKG2D receptor. DISCUSSION: Because MHC expression is often down-regulated on tumor cells and the NKG2D ligands are generally specific to malignant cells, the adoptive transfer of CEC that kill through different pathways may circumvent tumor-resistant mechanisms and improve outcomes.     

Requirement of species-specific interactions for the activation of human gamma delta T cells by pamidronate

Human gammadelta T cells bearing Vgamma2Vdelta2-TCR recognize various kinds of small nonpeptide Ags, and activation of them by a nitrogen-containing bisphosphonate Ag, pamidronate, requires Ag presentation by cells other than gammadelta T cells, including many human tumor cells. Present results demonstrated that tumor cell lines of nonhuman origins pulsed with pamidronate failed to activate human gammadelta T cells without exception, whereas most if not all human tumor cell lines could do so. Gammadelta T cells formed stable conjugates with pamidronate-pulsed human tumor cells and both conjugate formation and gammadelta T cell activation were inhibited significantly by anti-LFA-1 mAb, suggesting the requirement of LFA-1-mediated interaction with APC for efficient gammadelta T cell activation. Consistently, ICAM-1(low) tumor cell lines pulsed with pamidronate induced no or only weak activation of gammadelta T cells, whereas similarly treated ICAM-1(high) cell lines could activate them. One of the two ICAM-1(low) tumor cell lines pulsed with pamidronate induced strong gammadelta T cell activation after ICAM-1 gene transfer. However, another ICAM-1(low) human cell line as well as murine tumor cell lines pulsed with pamidronate remained totally defective in gammadelta T cell activation even after expression of human ICAM-1. These results suggested that activation of human gammadelta T cells by nonpeptide Ags required species-specific interactions in addition to LFA-1/ICAM-1-mediated cell adhesion with APC.     

Correlation between T cell receptor gamma delta isotypic forms and cytotoxic activity: analysis with human T cell clones and lines

Three biochemically distinct isotypic forms of the human T cell receptor (TcR) gamma delta structure can be expressed at the cell membrane. This unique variation in structure of TcR, which is due to C gamma gene segments utilization, prompted us to look for isotype-association functional differences. In this regard, we have developed human T cell clones or lines from normal thymus or peripheral blood from several patients. In the present report, we have selected by phenotypic, biochemical, and TcR gene rearrangement analysis representative pairs of IL2-dependent clones or lines for each TcR gamma delta isotypic form. The results showed a lack of correlation between the TcR isotypes and the ability of the cells to proliferate in response to TcR stimulation mediated through the CD3 molecular complexes. By contrast, despite the fact that all of these representative cells exhibit an NK-like activity, as measured by their ability to kill K562, the strongest lytic activity was observed with the cells having the disulfide-bonded form of the receptor. Moreover only those latter cells were able to efficiently kill the LAK-sensitive Daudi cell line.     

Differentiation of human CD8(+) T cells from a memory to memory/effector phenotype

Previous studies of perforin expression and cytokine production in subsets of peripheral human CD45RA(-)CD8(+) T cells with different CD28/CD27 phenotypes showed that CD28(+)CD45RA(-)CD8(+) and CD27(+)CD45RA(-)CD8(+) T cells have characteristics of memory T cells, whereas CD28(-)CD45RA(-)CD8(+) and CD27(-)CD45RA(-)CD8(+) T cells have characteristics of both memory and effector T cells. However, the differentiation pathway from memory CD8(+) T cells into memory/effector CD8(+) T cells has not been completely clarified. We investigated this differentiation pathway using EBV- and human CMV (HCMV)-specific CD8(+) T cells. Three subsets of CD45RA(-)CD8(+) T cells were observed in both total CD8(+) T cells and EBV- or HCMV-specific CD8(+) T cells: CD27(+)CD28(+), CD27(+)CD28(-), and CD27(-)CD28(-). A significant number of the CD27(-)CD28(+) subset was observed in total CD8 T cells. However, this subset was barely detectable in EBV- or HCMV-specific CD8(+) T cells. Analysis of perforin expression and cytotoxic activity in the first three subsets suggested the following differentiation pathway: CD27(+)CD28(+)CD45RA(-)-->CD27(+)CD28(-)CD45RA(-)-->CD27(-)CD28(-)CD45RA(-). This was supported by the observation that the frequency of CCR5(+) cells and CCR7(+) cells decreased during this sequence. Analysis of CCR5 and CCR7 expression in the CD27(+)CD28(+) memory cell subset demonstrated the presence of three CCR5/CCR7 populations: CCR5(-)CCR7(+), CCR5(+)CCR7(+), and CCR5(+)CCR7(-). These findings suggested the following differentiation pathway: CD27(+)CD28(+)CD45RA(-) (CCR5(-)CCR7(+)-->CCR5(+)CCR7(+)-->CCR5(+)CCR7(-))-->CD27(+)CD28(-)CD45RA(-)-->CD27(-)CD28(-)CD45RA(-). The presence of a CD27(-)CD28(+) subset with a CCR5(+)CCR7(-) phenotype implies a specialized role for this subset in the differentiation of CD8(+) T cells.     

Regulation of IL-17 in human CCR6+ effector memory T cells

IL-17-secreting T cells represent a distinct CD4(+) effector T cell lineage (Th17) that appears to be essential in the pathogenesis of numerous inflammatory and autoimmune diseases. Although extensively studied in the murine system, human Th17 cells have not been well characterized. In this study, we identify CD4(+)CD45RO(+)CCR7(-)CCR6(+) effector memory T cells as the principal IL-17-secreting T cells. Human Th17 cells have a unique cytokine profile because the majority coexpress TNF-alpha but not IL-6 and a minor subset express IL-17 with IL-22 or IL-17 and IFN-gamma. We demonstrate that the cytokines that promote the differentiation of human naive T cells into IL-17-secreting cells regulate IL-17 production by memory T cells. IL-1beta alone or in association with IL-23 and IL-6 markedly increase IL-17(+) CCR6(+) memory T cells and induce IL-17 production in CCR6(-) memory T cells. We also show that T cell activation induces Foxp3 expression in T cells and that the balance between the percentage of Foxp3(+) and IL-17(+) T cells is inversely influenced by the cytokine environment. These studies suggest that the cytokine environment may play a critical role in the expansion of memory T cells in chronic autoimmune diseases.     

Antigen presentation by an immature myeloid dendritic cell line does not cause CTL deletion in vivo, but generates CD8+ central memory-like T cells that can be rescued for full effector function

Immature dendritic cells (DC), in contrast to their mature counterparts, are incapable of mobilizing a CD8+ CTL response, and, instead, have been reported to induce CTL tolerance. We directly addressed the impact of immature vs mature DC on CTL responses by infusing adenovirus peptide-loaded DC (of the D1 cell line) into mice that had received adenovirus-specific naive TCR-transgenic CD8+ T cells. Whereas i.v. injection of mature DC triggered vigorous CTL expansion, immature DC elicited little proliferation involving only a minority of the TCR-transgenic CTL. Even though the latter CTL developed effector functions, including cytolytic activity and proinflammatory cytokine secretion, these cells differed significantly from CTL primed by mature DC in that they did not exhibit down-regulation of CD62L and CCR7, receptors involved in trapping of T cells in the lymphoid organs. Interestingly, adoptive transfer of CTL effector cells harvested after priming by either mature or immature DC into naive recipient mice, followed by exposure to adenovirus, yielded quantitatively and qualitatively indistinguishable CTL memory responses. Therefore, in vivo priming of naive CD8+ T cells by immature DC, although failing to induce a full-blown, systemic CTL response, resulted in the formation of central memory-like T cells that were able to expand and produce IFN-gamma upon secondary antigenic stimulation.     

Imaging effector functions of human cytotoxic CD4+ T cells specific for Plasmodium falciparum circumsporozoite protein

Malaria vaccines, comprised of irradiated Plasmodium falciparum sporozoites or a synthetic peptide containing T and B cell epitopes of the circumsporozoite protein (CSP), elicit multifunctional cytotoxic and non-cytotoxic CD4⁺ T cells in immunised volunteers. Both lytic and non-lytic CD4⁺T cell clones recognised a series of overlapping epitopes within a ‘universal’ T cell epitope EYLNKIQNSLSTEWSPCSVT of CSP (NF54 isolate) that was presented in the context of multiple DR molecules. Lytic activity directly correlated with T cell receptor (TCR) functional avidity as measured by stimulation indices and recognition of naturally occurring variant peptides. CD4⁺ T cell-mediated cytotoxicity was contact-dependent and did not require de novo synthesis of cytotoxic mediators, suggesting a granule-mediated mechanism. Live cell imaging of the interaction of effector and target cells demonstrated that CD4⁺ cytotoxic T cells recognise target cells with their leading edge, reorient their cytotoxic granules towards the zone of contact, and form a stable immunological synapse. CTL attacks induced chromatin condensation, nuclear fragmentation and formation of apoptotic bodies in target cells. Together, these findings suggest that CD4⁺ CTLs trigger target cell apoptosis via classical perforin/granzyme-mediated cytotoxicity, similar to CD8⁺ CTLs, and these multifunctional sporozoite- and peptide-induced CD4⁺ T cells have the potential to play a direct role as effector cells in targeting the exoerythrocytic forms within the liver.     

Immortalization of human T cells expressing T-cell receptor gamma delta by herpesvirus saimiri.

Herpesvirus saimiri (HVS) has recently been shown to immortalize human CD4+ and CD8+ T cells expressing T-cell receptor alpha beta (TCR-alpha beta) with the maintenance of their original phenotypes and functional properties. However, the immortalization of human T cells expressing TCR-gamma delta by HVS has not been successful. Here we report that HVS can also infect and immortalize human T cells expressing TCR-gamma delta. Two human TCR-gamma delta+ T-cell clones, which continuously proliferated in interleukin-2-containing culture medium without any exogenous stimulation or addition of feeder cells for more than 8 months, were established by HVS infection. Morphologically, the HVS-transformed TCR-gamma delta+ T-cell clones were granular lymphocytes which exhibited wide-range HLA-unrestricted cytotoxicity as untransformed TCR-gamma delta+ T cells. Their phenotypes and cytotoxic activities were not altered during long-term culture. The immortalization of human TCR-gamma delta+ T cells by HVS infection would be useful for functional analysis of this lymphocyte population, which is believed to play an important role in protection against various infectious diseases.     

Increased numbers of preexisting memory CD8 T cells and decreased T-bet expression can restrain terminal differentiation of secondary effector and memory CD8 T cells

Memory CD8 T cells acquire effector memory cell properties after reinfection and may reach terminally differentiated, senescent states ("Hayflick limit") after multiple infections. The signals controlling this process are not well understood, but we found that the degree of secondary effector and memory CD8 T cell differentiation was intimately linked to the amount of T-bet expressed upon reactivation and preexisting memory CD8 T cell number (i.e., primary memory CD8 T cell precursor frequency) present during secondary infection. Compared with naive cells, memory CD8 T cells were predisposed toward terminal effector (TE) cell differentiation because they could immediately respond to IL-12 and induce T-bet, even in the absence of Ag. TE cell formation after secondary (2°) or tertiary infections was dependent on increased T-bet expression because T-bet(+/-) cells were resistant to these phenotypic changes. Larger numbers of preexisting memory CD8 T cells limited the duration of 2° infection and the amount of IL-12 produced, and consequently, this reduced T-bet expression and the proportion of 2° TE CD8 T cells that formed. Together, these data show that over repeated infections, memory CD8 T cell quality and proliferative fitness is not strictly determined by the number of serial encounters with Ag or cell divisions, but is a function of the CD8 T cell differentiation state, which is genetically controlled in a T-bet-dependent manner. This differentiation state can be modulated by preexisting memory CD8 T cell number and the intensity of inflammation during reinfection. These results have important implications for vaccinations involving prime-boost strategies.