Prolonged antigen expression following DNA vaccination impairs effector CD8+ T cell function and memory development

After priming, naive T cells undergo a program of expansion, contraction, and memory formation. Numerous studies have indicated that only a brief period of antigenic stimulation is required to fully commit CD8+ T cells to this program. Nonetheless, the persistence of Ag may modulate the eventual fate of CD8+ T cells. Using DNA delivery, we showed previously that direct presentation primes high levels of effector CD8+ T cells as compared with cross-presentation. One explanation now revealed is that prolonged cross-presentation limits effector cell expansion and function. To analyze this, we used a drug-responsive system to regulate Ag expression after DNA injection. Reducing expression to a single burst expanded greater numbers of peptide-specific effector CD8+ T cells than sustained Ag. Consequences for memory development were assessed after boosting and showed that, although persistent Ag maintained higher numbers of tetramer-positive CD8+ T cells, these expanded less (approximately 4-fold) than those induced by transient Ag expression (approximately 35-fold). Transient expression at priming therefore led to a net higher secondary response. In terms of vaccine design, we propose that the most effective DNA-based CD8+ T cell vaccines will be those that deliver a short burst of Ag.     

Regulatory T cell expression of herpesvirus entry mediator suppresses the function of B and T lymphocyte attenuator-positive effector T cells

The binding of herpesvirus entry mediator (HVEM) to B and T lymphocyte attenuator (BTLA) is known to activate an inhibitory signaling cascade in effector T (Teff) cells, but we now report that the HVEM-BTLA pathway is also important to the suppressive function of regulatory T cells (Tregs). Although naive T cells up-regulated BTLA upon TCR activation, Treg expression of BTLA remained low, regardless of TCR activation. Moreover, BTLA(-/-) CD4(+)CD25(+) Tregs had normal suppressive activity, whereas BTLA(-/-) Teff cells were more resistant than wild-type Teff cells to suppression by Tregs, suggesting BTLA expression by Teff cells was required for their suppression by Tregs. In contrast to BTLA, HVEM expression was comparable in naive Tregs vs Teff cells, but after stimulation HVEM expression was quickly down-regulated by Teff cells, whereas HVEM was further up-regulated by Tregs. HVEM(-/-) Tregs had decreased suppressive activity as compared with wild-type Tregs, indicating that Treg expression of HVEM was required for optimal suppression. Consistent with this, T cells from Scurfy mice (FoxP3 mutant) lacked HVEM gene expression, and adoptively transferred wild-type but not HVEM(-/-) Tregs were able to control alloresponses in vivo by normal Teff cells. Our data demonstrate that Tregs can exert their effects via up-regulation of the negative costimulatory ligand HVEM, which upon binding to BTLA expressed by Teff cells helps mediate the suppressive functions of Tregs in vitro and in vivo.     

PLC gamma 1, a possible mediator of T cell receptor function

Stimulation of T cell antigen receptor (TCR/CD3) following the recognition of peptide-major histocompatibility antigen complex induces phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. However, the phospholipase C (PLC) enzyme mediating this process has not been identified. We report that PLC gamma 1 protein is expressed in human T cells. It is a phosphoprotein, and the activation of cyclic AMP-dependent protein kinase (PKA) or of protein kinase C (PKC) with forskolin or phorbol ester, respectively, increases the level of phosphorylation. CD3 stimulation of T cells induces tyrosine phosphorylation of PLC gamma 1 and causes 8-10-fold higher yield of PLC activity with anti-phosphotyrosine antibody (APTyr Ab) from activated cells than from non-activated cells. Genistein, an inhibitor of protein tyrosine kinase, decreases this yield of AP-Tyr Ab-bound PLC activity from activated cells and lowers the level of Ca2+ mobilization. Furthermore, phorbol ester and forskolin treatment of cells before CD3 stimulation reduces the level of tyrosine phosphorylation of PLC gamma 1 and the PLC activity associated with APTyr Ab. These results suggest that CD3 stimulation activates PIP2 hydrolysis by inducing tyrosine phosphorylation of PLC gamma 1, which is regulated negatively by PKC and PKA.     

IL-1 and IL-4 modulate IL-1 receptor expression in a murine T cell line

The combination of IL-1 and IL-4 stimulates the proliferation of certain murine T cell populations. Although this effect has been best characterized for a number of murine type 2 Th cell (Th2) clones, the mechanism(s) by which these cytokines effect this response is unclear. We have examined the effects of IL-1 and IL-4 on IL-1R expression by MD10 cells, and IL-1-responsive murine T cell line. These cells bear specific IL-1R, which bind human and murine IL-1 alpha and -beta. The measured apparent IL-1R dissociation constant ranged from 41 to 255 pM using 125I-HrIL-1 alpha. Cross-linking studies demonstrated two different 125I-HrIL-1 alpha binding complexes having Mr of 70,000 and 130,000 to 156,000. When removed from passage conditions and placed in non-growth factor-supplemented media, MD10 IL-1R expression spontaneously increased two- to fourfold over the first 11 to 12 h of culture followed by a decline. This phenomenon is partially inhibitable by cycloheximide suggesting that protein synthesis is involved. In agreement with other reports, HrIL-1 alpha down-regulated the expression of its own receptor with an ED50 of between 1 and 10 pM HrIL-1 alpha for this effect. In most experiments, low amounts of HrIL-1 alpha (1.0, 0.1 pM) significantly augmented IL-1R expression. Scatchard analysis of data obtained with all HrIL-1 alpha treatment conditions showed that the effects were due to a change in receptor number, not affinity. Significantly, purified murine IL-4 (MpIL-4) augmented MD10 IL-1R expression in both a time- and dose-dependent fashion. In the presence of 50 U/ml MpIL-4, MD10 IL-1R expression increased two- to threefold after 24 h without a change in receptor affinity. When MpIL-4 (50 U/ml) and various amounts of HrIL-1 alpha (.01-1000 pM) were co-added, the down-regulatory effect of high levels of HrIL-1 alpha was significantly antagonized. When added to cultures after 24 h of HrIL-1 alpha (100 pM) treatment, MpIL-4 reversed the IL-1R down-regulatory effect induced by high levels of HrIL-1 alpha. Finally, when combined in MD10 proliferation assays, MpIL-4 synergistically enhanced the proliferation of MD10 cells treated with suboptimal levels of HrIL-1 alpha.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro induction and expression of interleukin 2 receptor in a clonal T helper cell differentiation model

Induction and expression of interleukin 2 (IL 2) receptor have been studied using a poly( Glu60 Ala30 Tyr10 ) (GAT)-specific T cell clone of mouse origin. This clone (52-3) has been characterized and it exhibits functional properties of T helper (TH) cells: it leads to a specific anti-DNP response in the presence of DNP-GAT and DNP-primed B cells and it secretes biological activities which can induce polyclonal B cell proliferation and IgM secretion. In vitro this clone mimics the activation stages of normal T lymphocytes and can be obtained under two states of differentiation. depending on the antigen-specific signal provided by antigen-presenting cells (APC). The expression of IL 2 receptor by this clone has been studied by (i) its response to IL 2, (ii) its capacity to absorb IL 2 bioactivity, and (iii) its reactivity with monoclonal antibody 7D4 specific for mouse IL 2 receptor. All the results indicate that the unstimulated state does not express the IL 2 receptor while the activated state does. Clone 52-3 has been compared with clone 14-1.6 that derives from a TH cell line and expresses the IL 2 receptor constitutively. 52-3 offers a good experimental model for studying in vitro, in a clonal TH cell population, the detailed mechanism of IL 2 receptor induction.     

Myeloid-derived suppressor cells accumulate in kidney allograft tolerance and specifically suppress effector T cell expansion

The immune tolerance to rat kidney allografts induced by a perioperative treatment with anti-CD28 Abs is associated with a severe unresponsiveness of peripheral blood cells to donor Ags. In this model, we identified an accumulation in the blood of CD3(-)class II(-)CD11b(+)CD80/86(+) plastic-adherent cells that additionally expressed CD172a as well as other myeloid markers. These cells were able to inhibit proliferation, but not activation, of effector T cells and to induce apoptosis in a contact-dependent manner. Their suppressive action was found to be under the control of inducible NO synthase, an enzyme also up-regulated in tolerated allografts. Based on these features, these cells can be defined as myeloid-derived suppressor cells (MDSC). Interestingly, CD4(+)CD25(high)FoxP3(+) regulatory T cells were insensitive in vitro to MDSC-mediated suppression. Although the adoptive transfer of MDSC failed to induce kidney allograft tolerance in recently transplanted recipients, the maintenance of tolerance after administration of anti-CD28 Abs was found to be dependent on the action of inducible NO synthase. These results suggest that increased numbers of MDSC can inhibit alloreactive T cell proliferation in vivo and that these cells may participate in the NO-dependent maintenance phase of tolerance.     

Dynamics and requirements of T cell clonal expansion in vivo at the single-cell level: effector function is linked to proliferative capacity

The adoptive transfer of TCR-transgenic T cells into syngeneic recipients allows characterization of individual T cells during in vivo immune responses. However, the proliferative behavior of individual T cells and its relationship to effector and memory function has been difficult to define. Here, we used a fluorescent dye to dissect and quantify T cell proliferative dynamics in vivo. We find that the average Ag-specific CD4+ T cell that undergoes division in vivo generates >20 daughter cells. TCR and CD28 signals cooperatively determine the degree of primary clonal expansion by increasing both the proportion of Ag-specific T cells that divide and the number of rounds of division the responding T cells undergo. Nonetheless, despite optimal signaling, up to one-third of Ag-specific cells fail to divide even though they show phenotypic evidence of Ag encounter. Surprisingly, however, transgenic T cells maturing on a RAG-2-/- background exhibit a responder frequency of 95-98% in vivo, suggesting that maximal proliferative potential requires either a naive phenotype or allelic exclusion at the TCRalpha locus. Finally, studies reveal division cycle-dependent expression of markers of T cell differentiation, such as CD44, CD45RB, and CD62L, and show also that expression of the cytokines IFN-gamma and IL-2 depends primarily on cell division rather than on receipt of costimulatory signals. These results provide a quantitative assessment of T cell proliferation in vivo and define the relationship between cell division and other parameters of the immune response including cytokine production, the availability of costimulation, and the capacity for memory.     

Preservation of immune effector cell function following administration of a dose-intense 5-fluorouracil-chemotherapy regimen

In a phase II clinical trial of 5-fluorouracil (5FU) plusN-(phosphonacetyl)-l-aspartate (PALA) therapy administration, a number of slowly developing clinical responses were observed. Because of this, a variety of immune parameters were sequentially studied in 21 patients on this trial. Of the 21 patients studied, 20 provided sufficient samples to compare baseline with subsequent values, 10 of the 20 patients responded to treatment. Responders and non-responders did not differ in any studied parameter at baseline. After 2 months of therapy, non-specific monocyte cytotoxicity (NSMC), antibody-dependent monocyte cytotoxicity (ADMC) and natural killer (NK) activity were higher in the entire study population, but these increases were not statistically significant. When responders and non-responders were evaluated separately, it was apparent that the trend was due solely to the changes observed in the responding patient population. When mean lysis values for each patient group were determined for each studied time point, it was possible to generate a mean area under the cytotoxicity/time curve (AUC) for each studied parameter. NSMC and ADMC did not differ in responders and non-responders. However, NK activity was significantly greater by mean AUC analysis (P = 0.006) in the responding group; NK activity was maintained in the responders, but decreased in non-responders. When lymphocyte and monocyte expression of the surface markers ₂-microglobulin, HLA-DR, CD56, HNK-1, CD 16 and interleukin-2 receptor were evaluated, there were no differences among responders and non-responders at baseline by mean AUC analysis or when comparing baseline with non-base-line values. It is concluded that although baseline immunological characteristics do not identify patients who are likely to respond to weekly 5FU and PALA, treatment is not associated with deleterious effects on the immune effector function parameters evaluated in this study, there being no effects on expression of a variety of associated cell-surface molecules.     

4-1BB ligand-mediated costimulation of human T cells induces CD4 and CD8 T cell expansion, cytokine production, and the development of cytolytic effector function

4-1BB (CD137) is a costimulatory member of the TNFR family expressed on activated T cells. Its ligand, 4-1BBL, is expressed on activated APC. In the mouse, CD8 T cells are preferentially activated by agonistic anti-murine 4-1BB Abs. However, murine 4-1BBL can stimulate both CD4 and CD8 T cells. To date, there are only limited data on the effects of 4-1BBL on human T cell responses. To further understand the role of 4-1BBL in human T cell responses, we compared human CD4 and CD8 T cell responses to transfected human 4-1BBL plus TCR-mediated stimulation. Both human CD4 and CD8 T cells responded to 4-1BBL. The presence of 4-1BBL on the APC led to increased expansion, cytokine production, and the development of cytolytic effector function by human T cells. In unfractionated T cell cultures, CD4 and CD8 T cells could expand to a similar extent in response to signals through the TCR and 4-1BB, as measured by CFSE labeling and by quantitating T cell numbers in the cultures. In contrast to the results with total T cells, isolated CD8 T cells produced less IL-2 and expanded to a lesser extent than isolated CD4 T cells responding to 4-1BBL. Thus, 4-1BBL is most effective when both CD4 and CD8 T cells are included in the cultures. CD28 and 4-1BB were found to synergize in the induction of IL-2 by human T cells, and CTLA-Ig partially blocked 4-1BBL-dependent IL-2 production. However, a portion of the 4-1BBL-mediated effects were independent of CD28-B7 interaction.     

Preferential expression of a common T cell receptor structure by T cells induced to proliferate in vitro with a murine retrovirus.

We have successfully isolated continuous T cell lines from murine spleen which have been induced to proliferate after in vitro exposure to the murine leukemia virus RadLV. Cell lines isolated from several strains of mice have an "immature" phenotype and are immortalized CD4- CD8- CD3+ cell lines. Cell lines of similar phenotype have now been derived from many individual mice, after spleens have been infected with two different RadLV viruses, a leukemogenic and a nonleukemogenic isolate. Among cell lines induced with RadLV/C6VL, an unusually high proportion of cells was found to bind the 124-40 anticlonotypic antibody specific for the alpha beta TCR expressed by C6VL/1 cells which produces RadLV/C6VL. This was not reflected in cell lines induced with the RadLV/V13 isolate nor in various lymphocyte subsets freshly isolated from normal mice, or induced to proliferate in culture. Cells expressing a common TCR structure would appear to be appropriate targets for in vitro proliferation and transformation induced by RadLV.     

1,25-Dihydroxyvitamin D3 increases type 1 interleukin-1 receptor expression in a murine T cell line

The biologically active metabolite of vitamin D₃, 1,25 (OH)₂ D₃, exerts important immunoregulatory effects in addition to being a central mediator of calcium/phosphate metabolism. Utilizing an interleukin 1 responsive murine T cell line and ¹²⁵I-interleukin 1α, we show that 1,25 (OH)₂ D₃ (5,50 nM) enhanced ¹²⁵I-interleukin 1α binding up to almost 2-fold over control. This 1,25 (OH)₂ D₃ effect occurred in a dose-dependent manner and was detectable after 24 h but not before 7 h of culture. Scatchard analysis of ¹²⁵I-interleukin 1α binding data demonstrated that 1,25 (OH)₂ D₃ enhanced interleukin 1 receptor number without a significant change in affinity. The biologically less potent metabolite of vitamin D₃, 25 (OH) D₃, also augmented ¹²⁵I-interleukin 1α binding but at steroid levels 2–3 log orders greater than 1,25 (OH)₂ D₃. This observation, combined with the presence of high-affinity ³H-1,25 (OH)₂ D₃ receptors (88 sites/cell, K = 0.45 nM) in cytosolic extracts, strongly suggests that the nuclear vitamin D receptor mediates this steroid's effect on interleukin 1 receptor expression. Based on the capacity of an anti-type 1 interleukin 1 receptor monoclonal antibody (35F5) to block 1,25 (OH)₂ D₃-enhanced ¹²⁵I-interleukin 1α binding, we conclude that this steroid augments type 1 interleukin 1 receptor expression. When combined with interleukin 1, a cytokine that also impacts MD10 interleukin 1 receptor expression, 1,25 (OH)₂ D₃ enhanced interleukin 1 receptor expression. Northern blots hybridized with a ³²P-type 1 interleukin 1 receptor cDNA probe show that 1,25 (OH)₂ D₃ enhanced type 1 interleukin 1 receptor steady state mRNA levels. Functionally, 1,25 (OH)₂ D₃ pretreatment augmented the MD10 proliferative response to suboptimal levels of interleukin 1 (< 100 fM interleukin 1α). These findings further support 1,25 (OH)₂ D₃'s role as an immunoregulatory molecule and provides a possible mechanism by which this steroid could potentiate certain immune activities.     

Cutting edge: expression of chemokine receptor CXCR1 on human effector CD8+ T cells

IL-8 is a potent inflammatory cytokine that induces chemotaxis of neutrophils expressing CXCR1 and CXCR2, thus indicating its involvement in the migration of these cells to inflammatory sites where bacteria proliferate. Presently, we showed that CXCR1(+) cells were predominantly found among CD8(+) T cells having effector phenotype, and that the expression of CXCR1 was positively correlated with that of perforin, suggesting that CXCR1 is expressed on effector CD8(+) T cells. Indeed, human CMV-specific CD8(+) T cells from healthy individuals, which mostly express the effector phenotype and have cytolytic function, expressed CXCR1, whereas EBV-specific CD8(+) T cells, which mostly express the memory phenotype and have no cytolytic function, did not express this receptor. The results of a chemotaxis assay showed that the migration of CXCR1(+)CD8(+) T cells was induced by IL-8. These results suggest that the IL-8-CXCR1 pathway plays an important role in the homing of effector CD8(+) T cells.     

Inhibition of effector function but not T cell activation and increase in FoxP3 expression in T cells differentiated in the presence of PP14

Background

T-helper polarization of naïve T cells is determined by a complex mechanism that involves many factors, eventually leading to activation of Th1, Th2, or Th17 responses or alternatively the generation of regulatory T cells. Placental Protein 14 (PP14) is a 28 kDa glycoprotein highly secreted in early pregnancy that is able to desensitize T cell receptor (TCR) signaling and modulate T cell activation.

Methodology/Principal Findings

Prolonged antigen-specific stimulation of T cells in the presence of PP14 resulted in an impaired secretion of IFN-γ, IL-5 and IL-17 upon restimulation, although the cells proliferated and expressed activation markers. Furthermore, the generation of regulatory CD4⁺CD25^highFoxp3⁺ T cells was induced in the presence of PP14, in both antigen-specific as well as polyclonal stimulation. In accordance with previous reports, we found that the induction of FoxP3 expression by PP14 is accompanied by down regulation of the PI3K-mTOR signaling pathway.

Conclusions/Significance

These data suggest that PP14 arrests T cells in a unique activated state that is not accompanied with the acquisition of effector function, together with promoting the generation of regulatory T cells. Taken together, our results may elucidate the role of PP14 in supporting immune tolerance in pregnancy by reducing T cell effector functions along with augmenting Treg differentiation.     

Establishment of gp100 and MART-1/Melan-A-specific cytotoxic T lymphocyte clones using in vitro immunization against preselected highly immunogenic melanoma cell clones

The induction of an in vitro T cell response against tumour-associated antigens with subsequent expansion of the individual cytotoxic T lymphocyte (CTL) clones still is not routine and the only tumour-associated antigen that has been found to easily induce the establishment of CTL clones is the MART-1/Melan-A antigen. In this paper, we describe a new approach for in vitro immunization based on the use of preselected melanoma cell clones. The human melanoma cell subline FM3.P was cloned and the immunological properties of individual clones were compared. Melanoma cell clone FM3.29, having a high level of expression of melanoma differentiation antigens, as well as high levels of the HLA class I and class II antigens and adhesion molecules, was used for the establishment of a CTL line that was subsequently cloned. For optimization of the conditions of growth of established CTL clones, a particular melanoma subline FM3.D/40 was selected for supporting the proliferation of CTL clones. The majority of the established CTL clones recognized the melanoma-associated differentiation antigens gp100 and MART-1/Melan-A. Epitope analysis indicated that two different epitopes derived from gp100 (154-162 and 280-288) and a single epitope from MART-1/Melan-A (27 35) were recognized by these CTL clones. The gp100-specific CTL clones were found to be significantly more sensitive to the culture conditions than the MART-1/Melan-A-specific CTL clones. In addition, the presence of excess peptide in the culture medium induced autokilling of the gp100-specific, but not the MART-1/Melan-A-specific CTL clones. Taken together, these results demonstrate that, by careful preselection of melanoma cell lines and clones both for the induction of CTL line from patients' peripheral blood lymphocytes and subsequent cloning, it is possible to obtain a large number of stable CTL clones even against such an inherently "difficult" differentiation antigen as gp100.     

TNF receptor 1 deficiency increases regulatory T cell function in nonobese diabetic mice

TNF has been implicated in the pathogenesis of type 1 diabetes. When administered early in life, TNF accelerates and increases diabetes in NOD mice. However, when administered late, TNF decreases diabetes incidence and delays onset. TNFR1-deficient NOD mice were fully protected from diabetes and only showed mild peri-insulitis. To further dissect how TNFR1 deficiency affects type 1 diabetes, these mice were crossed to β cell-specific, highly diabetogenic TCR transgenic I-A(g7)-restricted NOD4.1 mice and Kd-restricted NOD8.3 mice. TNFR1-deficient NOD4.1 and NOD8.3 mice were protected from diabetes and had significantly less insulitis compared with wild type NOD4.1 and NOD8.3 controls. Diabetic NOD4.1 mice rejected TNFR1-deficient islet grafts as efficiently as control islets, confirming that TNFR1 signaling is not directly required for β cell destruction. Flow cytometric analysis showed a significant increase in the number of CD4(+)CD25(+)Foxp3(+) T regulatory cells in TNFR1-deficient mice. TNFR1-deficient T regulatory cells were functionally better at suppressing effector cells than were wild type T regulatory cells both in vitro and in vivo. This study suggests that blocking TNF signaling may be beneficial in increasing the function of T regulatory cells and suppression of type 1 diabetes.     

CD8 T cell clonal expansion and development of effector function require prolonged exposure to antigen, costimulation, and signal 3 cytokine

Full activation of naive CD8 T cells requires Ag, costimulation, and a third signal that can be provided by IL-12. Brief exposure (6 h) to Ag and B7-1 is sufficient to stimulate multiple rounds of cell division, but clonal expansion and development of effector function are minimal even when signal 3 is present. Full activation instead requires concerted signaling by Ag, B7-1, and IL-12 for greater than 40 h. Thus, the gene expression program required for cell division can be initiated by brief interaction with Ag and costimulation, but maintaining the expression of the genes needed for survival and effector function requires prolonged signaling by a signal 3 cytokine in concert with Ag and costimulation.