Secondary Proliferative Responses Research Articles

IL-2 production by activated naïve and effector CD8 T cells forecasts memory formation. (LYM5P.700)

Abstract The clearance and control of many intracellular pathogens and tumors rely on the development of efficacious CD8 T cell responses. These responses are functionally diverse, however, whether and how this heterogeneity forecasts effector formation, memory potential, and protective capacity has not yet been definitively determined. To address this, we utilized IL-2 and IFN-γ double cytokine reporter mice to separate viable IL-2 and IFN-γ producing CD8 T cell subsets, and performed a series of adoptive transfer experiments to track their memory potential and fate. We discovered that within hours of activation, naïve CD8 T cells undergo a burst of cytokine synthesis and distinct populations develop that produce only IL-2, only IFN-γ, co-produce IL-2 and IFN-γ, or do not produce either cytokine. This diversity is consequential as newly activated naïve CD8 T cells that fail to produce IL-2 preferentially attain effector features. Conversely, autocrine IL-2 production by recently activated naïve CD8 T cells is associated with the formation of memory traits. Similarly, the ability of CD8 T cells to produce IL-2 during the effector phase also signified memory potential and the ability to mount vigorous secondary proliferative responses. Together these data demonstrate that functional heterogeneity influences the differentiation of CD8 T cell responses and that effector and memory formation can be predicted by the pattern of autocrine IL-2 production.

Thymus‐resident memory CD8+T cells mediate local immunity

The thymus is a primary lymphoid organ responsible for production and selection of T cells. Nonetheless, mature T cells and in particular activated T cells can reenter the thymus. Here, we identified memory CD8(+) T cells specific for lymphocytic choriomeningitis virus or vaccinia virus in the thymus of mice long-time after the infection. CD8(+) T cells were mainly located in the thymic medulla, but also in the cortical areas. Interestingly, virus-specific memory CD8(+) T cells in the thymus expressed the cell surface markers CD69 and CD103 that are characteristic of tissue-resident memory T cells in a time-dependent manner. Kinetic analyses and selective depletion of peripheral CD8(+) T cells by antibodies further revealed that thymic virus-specific memory CD8(+) T cells did not belong to the circulating pool of lymphocytes. Finally, we demonstrate that these thymus-resident virus-specific memory CD8(+) T cells efficiently mounted a secondary proliferative response, exhibited immediate effector functions and were able to protect the thymus from lymphocytic choriomeningitis virus reinfection. In conclusion, the present study not only describes for the first time virus-specific memory CD8(+) T cells with characteristics of tissue-resident memory T (T(RM)) cells in a primary lymphoid organ but also extends our knowledge about local T-cell immunity in the thymus.

Instant Recall: A Key Role for Effector-Phenotype CD8+ Memory T Cells in Immune Protection

In this issue of Immunity, Olson et al. (2013) demonstrate that circulating CD8⁺ memory T cells with an effector-like phenotype, previously thought to be mostly senescent, provide robust protection from a secondary pathogen challenge despite their poor secondary proliferative response.

Intracranial atherosclerotic disease associated with moyamoya collateral formation: histopathological findings

Atherosclerotic disease has been suspected as a cause of moyamoya disease in some patients but has not, to the authors' knowledge, been confirmed by pathological studies. The authors present the histopathological findings in a patient with moyamoya collateral formation associated with atherosclerotic occlusive disease of the distal internal carotid artery (ICA). Typical atheromatous changes were evident in the distal ICA and proximal middle cerebral artery. In addition, intimal thickening, fibrosis, and abnormal internal elastic lamina were present in these vessels. These findings are common in moyamoya but not in atherosclerotic disease. Proliferation and enlargement of the lenticulostriate arteries in the basal ganglia was also identified. Moyamoya phenomenon secondary to atherosclerotic disease has similar histopathological features to idiopathic moyamoya phenomenon, both in the affected large basal arteries and lenticulostriate collaterals. These findings support the hypothesis advanced by Peerless that moyamoya is a 2-step process involving an obliterative vasculopathy of the terminal ICA and a secondary proliferative response.

Nab2 regulates secondary CD8+ T-cell responses through control of TRAIL expression

AbstractCD4+ Th cells are pivotal for the generation and maintenance of CD8+ T-cell responses. “Helped” CD8+ T cells receive signals during priming that prevent the induction of the proapoptotic molecule TNF-related apoptosis-inducing ligand (TRAIL) during reactivation, thereby enabling robust secondary expansion. Conversely, “helpless” CD8+ T cells primed in the absence of Th induce TRAIL expression after restimulation and undergo activation-induced cell death. In the present study, we investigated the molecular basis for the differential regulation of TRAIL in helped versus helpless CD8+ T cells by comparing their transcriptional profiles, and have identified a transcriptional corepressor, NGFI-A binding protein 2 (Nab2), that is selectively induced in helped CD8+ T cells. Enforced expression of Nab2 prevents TRAIL induction after restimulation of primary helpless CD8+ T cells, and expression of a dominant-negative form of Nab2 in helped CD8+ T cells impairs their secondary proliferative response that is reversible by TRAIL blockade. Finally, we observe that the CD8+ T-cell autocrine growth factor IL-2 coordinately increases Nab2 expression and decreases TRAIL expression. These findings identify Nab2 as a mediator of Th-dependent CD8+ T-cell memory responses through the regulation of TRAIL and the promotion of secondary expansion, and suggest a mechanism through which this operates.

Selective blockade of NF‐κB by novel mutated IκBα suppresses CD3/CD28‐induced activation of memory CD4+ T cells in asthma

Nuclear factor kappaB (NF-kappaB) overactivation plays a crucial role in T-helper 2 (Th2)-biased allergic airway inflammation by increased activation and decreased apoptosis of CD4(+) T cells. We have shown that targeted NF-kappaB suppression in dendritic cells by adenoviral gene transfer of a novel mutated inhibitor of NF-kappaB (IkappaBalpha) (AdIkappaBalphaM) contributes to T-cell tolerance, but the immunosuppressive action of AdIkappaBalphaM on memory (CD45RO(+)) CD4(+) T cells remains enigmatic. CD45RO(+) T cells from Dermatophagoides farinaei-sensitized asthmatic patients, untransfected or transfected with AdIkappaBalphaM or AdLacZ (beta-galactosidase) for 24 h, were stimulated with anti-CD3 (1.0 microg/ml) plus anti-CD28 (0.5 microg/ml) monoclonal antibody for an additional 24 h. IkappaBalphaM transgene expression and NF-kappaB activation were detected by polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR), Western blot analysis, and electrophoretic mobility shift assay. Phenotype and apoptosis were measured by flow cytometry, annexin V binding, and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling analyses. Cytokine production and cell proliferation were determined using enzyme-linked immunosorbent assay and [(3)H] thymidine incorporation. A unique 801-bp IkappaBalphaM cDNA and a dose-dependent increase in IkappaBalphaM transgene expression were observed in AdIkappaBalphaM-transfected CD45RO(+) T cells. Significantly, AdIkappaBalphaM inhibited CD3/CD28-mediated NF-kappaB activation in CD45RO(+) T cells, leading to evident apoptosis, reduction of eotaxin, RANTES, Th1 [interferon (IFN)-gamma and interleukin (IL)-2], and Th2 (IL-4, IL-5, and IL-13 despite a slight decrease in IL-10) cytokines and secondary proliferative response. AdIkappaBalphaM also upregulated cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and downregulated CD69 besides no change in CD28. IkappaBalphaM might be beneficial to augment memory CD4(+) T-cell tolerance through modulating B7-CD28/CTLA-4 co-stimulatory pathways and NF-kappaB-dependent cytokine profiles in allergic inflammatory diseases including asthma.

PO13-334 REGRESSION OF NORMOLIPEMIC XANTHELASMA PALPEBRARUM ASSOCIATED WITH INFLAMMATORY STATE IN CASTLEMAN'S DISEASE

PO13-334 REGRESSION OF NORMOLIPEMIC XANTHELASMA PALPEBRARUM ASSOCIATED WITH INFLAMMATORY STATE IN CASTLEMAN'S DISEASE

Suppression of established experimental autoimmune uveitis by anti-LFA-1alpha Ab.

To identify costimulatory molecules that are important in the effector phase of experimental autoimmune uveitis (EAU). EAU was induced in C57BL/6 (B6) mice by transfer of activated T cells specific for the interphotoreceptor-binding protein (IRBP) 1-20 peptide. The animals were then treated with and without anti-leukocyte function associated antigen (LFA)-1alpha mAb, at day 0 or 10 (disease onset) after T-cell transfer. Clinical signs of inflammation, ocular histology, and infiltrated inflammatory cells in the eye were compared. The primary and secondary proliferative responses of uveitogenic CD4 and CD8 T cells were tested after treatment with costimulatory blockers in vivo and in vitro. Moreover, the abilities of uveitogenic T cell trafficking and their interaction with retinal astrocytes were examined. Anti-LFA-1alpha Abs caused significant suppression of disease when administered either at the time of effector uveitogenic T cell transfer or at disease onset. Studies of the mechanisms by which anti-LFA-1alpha Ab inhibits the effector phase of uveitis demonstrated that it blocks multiple pathogenic events of uveitis mediated by IRBP-specific uveitogenic T cells, including the activation of T cells outside and inside the eye and the trafficking of activated autoreactive T cells into the inflammatory site. In addition, Ab treatment selectively suppressed the activation and expansion of pathogenic, but not regulatory, T cells in vivo. Anti-LFA-1alpha Abs are potent inhibitors of established autoimmune uveitis and that such treatment may be applicable not only for the prevention, but also the treatment, of T-cell-mediated autoimmune diseases.

A CD8 DE loop peptide analog prevents graft-versus-host disease in a multiple minor histocompatibility antigen-mismatched bone marrow transplantation model

Donor CD8(+) T cells can be potent mediators of graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation to either major histocompatibility complex (MHC) class I-or multiple minor histocompatibility antigen-mismatched recipients. To develop small molecular inhibitors of CD8(+) T-cell activity, theoretical structural analysis of the human CD8 alpha molecule was previously used to identify potential functional surface epitopes that interact with the MHC class I molecule. The DE loop (p71-78) was identified as such a target region, and a panel of synthetic cyclized peptide mimics of this region were tested for their inhibitory effects on cytotoxic T lymphocyte activity in human cell-mediated lympholysis assays. Peptide 1109 (CKRLGDTFVC) was most effective at inhibiting specific target cell lysis. Accordingly, studies were conducted to determine whether there was sufficient cross-species homology in the DE loop region and its nonpolymorphic interactive site on the beta(2)-microglobulin domain of the MHC class I molecule to allow similar inhibition of murine CD8(+) cytotoxic T lymphocyte activity. On the basis of strong in vitro inhibitory activity of 1109 in the murine system, the capacity of the peptide to inhibit in vivo CD8(+) T-cell effector functions in skin and hematopoietic stem cell transplantation models was examined. In the C57BL/6 anti-bm1 skin allograft rejection model, across an MHC class I barrier, a single injection of 1109 at the time of transplantation significantly prolonged graft survival. Moreover, 1109 administered at the time of transplantation in the multiple minor histocompatibility antigen-disparate B10.BR-->CBA GVHD model significantly prolonged the survival of lethally irradiated mice that underwent transplantation with donor bone marrow cells and CD8(+) T cells. Histopathologic analysis confirmed that mice treated with the synthetic peptide exhibited diminution of epithelial target cell injury. Specificity of the peptide effect was evidenced by draining lymph node cells from B10.BR mice that had been challenged with CBA lymphocytes and simultaneously treated with 1109. These cells could not generate secondary proliferative responses in vitro upon stimulation with CBA splenocytes but could respond to third-party C57BL/6 stimulation. Thus, the 1109 peptide has potential application in the prevention of CD8-mediated GVHD development.

Induction of allograft tolerance through costimulatory blockade: first selection of drugs in vitro

The development of an in vitro assay predicting the chances of graft survival after treatment with immunoregulatory agents is a major topic in transplantation. Antibodies (Abs) interfering in the costimulatory pathway are promising candidates for the induction of tolerance. To evaluate these antibodies for clinical use studies non-human primates are the only feasible option due to species specificity of the antibodies. Peripheral blood mononuclear cells, isolated from a large panel of rhesus monkeys, were used in a unidirectional mixed lymphocyte reaction to evaluate the ability of antibodies blocking the costimulatory pathway, to affect both primary and secondary proliferative and cytolytic allospecific immune responses in vitro. These blocking antibodies were also used in protocols prolonging allograft survival in a life-supporting kidney allotransplant model in rhesus macaques. The ultimate aim is to establish a correlation between parameters obtained in vitro and the success of transplantation in vivo. The combination of anti-CD80 and anti-CD86 resulted in a complete abrogation of the primary alloresponse as measured in a proliferation assay. Adding anti-CD40 significantly reduced this inhibitory effect although the in vivo effects of this antibody have been shown to be beneficial. The secondary response was most prominently inhibited by the combination of anti-CD80/86. Paradoxically, anti-CD40 alone markedly inhibited the secondary proliferative response, but did not add to the inhibitory effect of the combination of anti-CD80/86. The cytolytic response was inhibited maximally only when CsA was added to the combination of anti-CD80/86. Treatment with monoclonal antibodies alone without immunosuppressive drugs was sufficient to maintain graft survival during the time of treatment in most animals. However, rejection was initiated as soon as the treatment ceased and no tolerance, resulting in long-term graft and patient survival, was established. The complete inhibition of primary alloresponses and the partial inhibition of secondary proliferative alloresponses correlate with prolonged graft survival during treatment, but have no predictive value for the success of tolerance induction for kidney allografts in rhesus monkeys.

Generation of human CD8 T regulatory cells by CD40 ligand-activated plasmacytoid dendritic cells.

Although CD8 T cell–mediated immunosuppression has been a well-known phenomenon during the last three decades, the nature of primary CD8 T suppressor cells and the mechanism underlying their generation remain enigmatic. We demonstrated that naive CD8 T cells primed with allogeneic CD40 ligand–activated plasmacytoid dendritic cells (DC)2 differentiated into CD8 T cells that displayed poor secondary proliferative and cytolytic responses. By contrast, naive CD8 T cells primed with allogeneic CD40 ligand–activated monocyte-derived DCs (DC1) differentiated into CD8 T cells, which proliferated to secondary stimulation and killed allogeneic target cells. Unlike DC1-primed CD8 T cells that produced large amounts of interferon (IFN)-γ upon restimulation, DC2-primed CD8 T cells produced significant amounts of interleukin (IL)-10, low IFN-γ, and no IL-4, IL-5, nor transforming growth factor (TGF)-β. The addition of anti–IL-10–neutralizing monoclonal antibodies during DC2 and CD8 T cell coculture, completely blocked the generation of IL-10–producing anergic CD8 T cells. IL-10–producing CD8 T cells strongly inhibit the allospecific proliferation of naive CD8 T cells to monocytes, and mature and immature DCs. This inhibition was mediated by IL-10, but not by TGF-β. IL-10–producing CD8 T cells could inhibit the bystander proliferation of naive CD8 T cells, provided that they were restimulated nearby to produce IL-10. IL-10–producing CD8 T cells could not inhibit the proliferation of DC1-preactivated effector T cells. This study demonstrates that IL-10–producing CD8 T cells are regulatory T cells, which provides a cellular basis for the phenomenon of CD8 T cell–mediated immunosuppression and suggests a role for plasmacytoid DC2 in immunological tolerance.

Elevated CTLA-4 expression on CD4 T cells from periodontitis patients stimulated with Porphyromonas gingivalis outer membrane antigen.

To characterize the T cell response to Porphyromonas gingivalis, we examined the expression of costimulatory molecules on T cells derived from adult periodontitis patients with high serum antibody titre to P. gingivalis. The expression of CD28, CTLA-4, CD40 ligand (CD40L) on CD4+ T cells was analysed by flow cytometry. IL-10 and transforming growth factor-beta (TGF-beta) mRNA expression were determined by reverse transcription-polymerase chain reaction (RT-PCR) and subsequent image analysis. Peripheral blood mononuclear cells (PBMC) derived from periodontitis patients showed higher proliferative responses to P. gingivalis outer membrane (OM) than those from healthy controls (P < 0.05). The percentage of CTLA-4+ cells within CD4+ T cells of patients was significantly higher than that of healthy controls after P. gingivalis OM stimulation (33.0% versus 11.9%, P < 0.01). There was no significant difference in the percentages of CD28+ cells and CD40L+ cells, and the percentage of CD40L+ cells was low in both groups even after stimulation. Stimulation of PBMC with P. gingivalis OM induced significantly higher IL-10 mRNA expression in periodontitis patients than in healthy controls (P < 0.05). The level of TGF-beta mRNA expression of patients tended to be higher than that of healthy controls, but there was no significant difference. To elucidate the functional role of CTLA-4, we further investigated the secondary proliferative response to P. gingivalis OM. Interestingly, P. gingivalis OM stimulation did not enhance antigen-specific secondary response. Anti-CTLA-4 MoAb had no effect on proliferation in the presence of P. gingivalis OM. CTLA-4Ig suppressed the proliferative response significantly (P < 0.01). These results suggest that T cell responses to P. gingivalis OM may be regulated by CTLA-4 that is expressed at the late phase of T cell activation, and, in part, immunosuppressive cytokines. Taken together, CTLA-4 may play a crucial role in the pathogenesis of chronic inflammatory periodontal disease.

Tumor Gangliosides Inhibit the Tumor-Specific Immune Response

Tumor gangliosides are highly immunosuppressive membrane glycosphingolipids that are shed into the tumor cell microenvironment. We directly tested the impact of shed gangliosides on the in vivo antitumor immune response in a syngeneic fully autochthonous system (FBL-3 erythroleukemia cells, C57BL/6 mice, and highly purified FBL-3 cell gangliosides). The major FBL-3 ganglioside was identified as GM1b by mass spectrometry. Substantial ganglioside shedding (90 pmol/108 cells/h), a requisite for their inhibition of the immune function of tumor-infiltrating leukocytes, was detected. Immunosuppression by FBL-3 gangliosides was potent; 5-20 microM inhibited the tumor-specific secondary proliferative response (80-100%) and suppressed the generation of tumor-specific CTLs (97% reduction of FBL-3 cell lysis at an E:T ratio of 100:1). In vivo, coinjection of 10 nmol of FBL-3 gangliosides with a primary FBL-3 cell immunization led to a reduced response to a secondary challenge (the increase in the draining popliteal lymph node mass, cell number, and lymphocyte thymidine incorporation were lowered by 70, 69, and 72%, respectively). Coinjection of gangliosides with a secondary tumor challenge led to a 61, 74, and 42% reduction of the increase in lymph node mass, cell number, and thymidine uptake and a 63-74% inhibition of the increase of draining lymph node T cells (CD3+), B cells (CD19+), and dendritic cells/macrophages (Mac-3+). Overall, the clear conclusion that tumor-derived gangliosides inhibit syngeneic antitumor immune responses implicates these molecules as a potent factor in promoting tumor formation and progression.

BALB/c Invariant Chain Mutant Mice Display Relatively Efficient Maturation of CD4+ T Cells in the Periphery and Secondary Proliferative Responses Elicited upon Peptide Challenge

Allelic differences are known to influence many important aspects of class II biosynthesis, including subunit assembly, Ii chain associations, and DM-mediated peptide loading. Mutant mouse strains lacking Ii chain expression have been previously studied on mixed genetic backgrounds. The present experiments describe cellular and functional characteristics of congenic BALB/c Ii chain mutants. As expected, class II surface expression was markedly decreased, but in contrast to I-Ad-transfected cell lines, serological analysis of BALB/c Ii chain-deficient spleen cells gave no evidence for discordant expression of class II conformational epitopes. Thus, we conclude that properly folded class II molecules are exported via the Ii chain-independent pathway. Functional assays demonstrate consistently superior peptide-loading capabilities, suggesting that these I-Ad molecules are empty or occupied by an easily displaced peptide(s). Defective B cell development was observed for three mutant strains established on diverse genetic backgrounds. Ii chain function is also essential for optimal class II surface expression by mature splenic dendritic cells. Surprisingly, we observe in BALB/c Ii chain mutants, relatively efficient maturation of CD4+ T cells in the periphery and secondary proliferative responses elicited upon peptide challenge. The milder phenotype displayed by BALB/c Ii chain mutants in comparison with class II functional defects previously described for mouse strains lacking Ii chain is likely to have an effect on disease susceptibility.

IL–10 Induces a Th2 Cell Tolerance in Allergic Asthma

Background: Interleukin (IL)–10 induces a long–term antigen–specific anergy in human CD4+ T cells. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from house dust mite (Dermatophagoides farinae, Der f)–sensitized asthmatic patients. PBMCs were stimulated with Der f antigen for 72 h immediately after purification or after 48 h of resting culture with medium, and IL–10 and IL–5 in the culture supernatant were measured. PBMCs were also stimulated with Der f antigen for 7 days either immediately after purification or after 48 h of resting culture, after which cells were collected. Secondary proliferative responses of these cells to stimulation for 3 days with Der f antigen and mitomycin C–treated PBMCs as antigen–presenting cells or with phorbol myristate acetate (PMA) plus calcium ionophore were investigated. Results: Stimulation of PBMCs with Der f antigen immediately after purification significantly increased the proliferative response and IL–5 production. Stimulation of PBMCs with Der f antigen after resting culture with medium alone for 48 h significantly decreased IL–5 production and markedly increased IL–10 production. Although activation of cells with Der f antigen immediately after purification significantly increased secondary proliferative responses, stimulation after 48 h of resting culture failed to increase secondary proliferative responses. However, proliferation recovered when cells were activated with PMA plus calcium ionophore. Conclusion: These results suggest that antigen–specific Th2 cells are anergized by IL–10 and that Th2 cell tolerance may suppress eosinophilic inflammation in allergic asthma.

Presence of Pentoxifylline During T Cell Priming Increases Clonal Frequencies in Secondary Proliferative Responses and Inhibits Apoptosis

Naive T cells appear to be primed by specific Ag to differentiate into either effectors or memory cells. We have been analyzing the factors involved in this differential commitment in the priming of alloresponsive human T cells in vitro and have shown that the presence of a phosphodiesterase inhibitor, pentoxifylline (POX), during priming results in a decrease in the primary response and enhancement in the secondary proliferative response. We now show that the POX-mediated effect can be mimicked by dibutyryl cAMP. The secondary response enhancement is due to the effects of POX on the T cells rather than the APCs, because even fixed APCs can prime T cells in the presence of POX. POX affects T cells directly by increasing clonal frequency rather than the burst size of the secondary responders. The known inhibition of IL-2 production by POX is not responsible for this effect, because exogenous IL-2 supplementation does not block it. The presence of POX during priming alters the outcome of T cell activation, resulting in a lower frequency of cells expressing IL-2R alpha (CD25) and a decrease in their subsequent apoptosis, and this antiapoptotic effect is consistent with the enhanced commitment of T cells to secondary responsiveness by POX.

Staphylococcal enterotoxin B-induced T-cell anergy is mediated by regulatory T cells.

Naive T cells mount a vigorous proliferative response to superantigen (SAg) stimulation in vivo. The proliferative response is followed by a partial deletion of responder T cells. Part of the deletion process has recently been attributed to the action of regulatory cytotoxic T cells that recognize major histocompatibility complex (MHC) class I-associated antigen receptor determinants on the target cell surface. Responder T cells that survived the SAg response were found to be incapable of generating a secondary proliferative response to a SAg challenge. We show here that this 'anergy' is enforced by CD8-positive regulatory suppressive T cells. These regulatory cells inhibit cell division of preactivated T cells but not the Sag response of naive T cells. Regulatory T cells are not generated in the presence of cyclosporin A and, once activated, become inactivated or deleted when restimulated in the presence of this immunosuppressive drug.

Induction of mucosal immunity against herpes simplex virus type 1 in the mouse protects against ocular infection and establishment of latency.

Immune responses were assessed after intranasal immunization of mice with a mixture of herpes simplex virus type 1 (HSV-1) glycoproteins with cholera toxin and its B subunit as adjuvant. Antigen-specific serum antibodies, which were largely IgG with IgG1 the major subclass, neutralized virus in vitro with a titer equivalent to that elicited by active infection. Significant levels of antigen-specific IgA were found in mucosal fluids of the eye as well as the vagina. Lymphocytes from draining lymph nodes showed secondary proliferative responses when cultured with HSV-1 in vitro, in immunized mice only, with the production of interleukin-2, interferon-gamma, interleukin-4, and interleukin-5. After ocular challenge, immunized mice were protected against the development of severe eye disease, zosteriform spread, or encephalitis, whereas the incidence of clinical symptoms in mock-immunized mice was 83%, 74%, and 52%, respectively. Finally, the incidence of latency was reduced from 88% to 13% after intranasal immunization.

Differential regulation of T cell activation for primary versus secondary proliferative responses.

T cell activation in vivo results in proliferation and generation of effector cytokine-secreting cells, as also in development of memory cells that mount enhanced responses upon restimulation. However, differences in the signals promoting generation of effector vs memory T cells are not yet characterized. In this study, using various strategies to modulate an allorecognition system for priming human T cells in vitro, we show that there are indeed differences between the signaling requirements for a first proliferative response and those for priming T cells for enhanced recall proliferative responses. Using APCs fixed with varying concentrations of paraformaldehyde, we show that the loss of ability of these APCs to generate a first response is not matched by a similar loss in their ability to prime responder T cells for recall responses. Prevention of DNA replication during T cell priming with aphidicolin, a DNA polymerase inhibitor, is not inimical to successful T cell priming. Thus, clonal expansion during priming is less crucial than the primed activation status of T cells for the enhanced recall response. We also show that pentoxifylline, a phosphodiesterase inhibitor, inhibits the primary proliferative response, but its presence during priming enhances the recall response capabilities of T cells. On the other hand, the presence of the calcineurin inhibitor cyclosporin A during priming reduces the efficiency of priming, but at low concentrations it induces, like pentoxifylline, enhancement in recall response capability. These findings have significant implications in designing immunosuppressive therapy and in the analysis of signals for T cell memory commitment.

Lymphoblastoid cells transfected with c-myc: downregulation of EBV-lytic antigens and impaired response of autologous CD4+ T cells in vitro.

Normal EBV-positive lymphoblastoid B-cell lines (LCL) were transfected with vectors containing the c-myc oncogene (pHEBO-E(mu)-myc) or control vectors (pHEBO-E(mu)) and analyzed for the expression of EBV-lytic and latent antigens. While EBV-latent antigens were normal in the c-myc transfectants, there was an almost complete downregulation of EBV-lytic antigens, including BZLF1, EA(D), gp340 and VCA. These observations were consistently repeated on 6 different LCLs transfected with c-myc. Unlike control LCLs, the c-myc transfectants did not release infectious EBV. PCR analysis demonstrated that BZLF1 mRNA was virtually absent in c-myc transfectants, possibly suggesting that the deregulated c-myc imposed a block in the EBV-lytic cycle at this particular level. c-myc transfectants failed to sustain the proliferative response of autologous CD4+ T-cell clones with specificity for EBV-lytic antigens. However, they regained this capacity after incubation with ultraviolet-inactivated EBV or gp340 antigen in vitro, also indicating that their antigen-presenting capacities were not impaired. c-myc transfectants failed to elicit a secondary proliferative response by autologous CD4+ T cells purified from the peripheral blood of EBV-seropositive donors. Exposure of c-myc transfectants to UV-inactivated EBV again resulted in a proliferative CD4+ T-cell response comparable to that elicited by the control LCLs. Collectively, our data provide evidence for the remarkable ability of an oncogene to influence the life cycle of a virus and to modify the antigenicity of the infected cells.