Acellular pertussis booster in adolescents induces Th1 and memory CD8+ T cell immune response

In a number of countries, whole cell pertussis vaccines (wcP) were replaced by acellular vaccines (aP) due to an improved reactogenicity profile. Pertussis immunization leads to specific antibody production with the help of CD4(+) T cells. In earlier studies in infants and young children, wcP vaccines selectively induced a Th1 dominated immune response, whereas aP vaccines led to a Th2 biased response. To obtain data on Th1 or Th2 dominance of the immune response in adolescents receiving an aP booster immunization after a wcP or aP primary immunization, we analyzed the concentration of Th1 (IL-2, TNF-α, INF-γ) and Th2 (IL-4, IL-5, IL-10) cytokines in supernatants of lymphocyte cultures specifically stimulated with pertussis antigens. We also investigated the presence of cytotoxic T cell responses against the facultative intracellular bacterium Bordetella pertussis by quantifying pertussis-specific CD8(+) T cell activation following the aP booster immunization. Here we show that the adolescent aP booster vaccination predominantly leads to a Th1 immune response based on IFNgamma secretion upon stimulation with pertussis antigen, irrespective of a prior whole cell or acellular primary vaccination. The vaccination also induces an increase in peripheral CD8(+)CD69(+) activated pertussis-specific memory T cells four weeks after vaccination. The Th1 bias of this immune response could play a role for the decreased local reactogenicity of this adolescent aP booster immunization when compared to the preceding childhood acellular pertussis booster. Pertussis-specific CD8(+) memory T cells may contribute to protection against clinical pertussis.     

Immunological memory is compromised by food restriction in deer mice Peromyscus maniculatus

The immune system protects organisms against infection, but this protection presumably comes at a cost. Here, we asked whether food restriction would compromise the ability of an organism to generate an immune response on reexposure to an antigen, which would represent a functional cost of immunological memory. Immunological memory is generated when B and T lymphocytes sensitive to components of pathogens (i.e., antigens) proliferate after exposure and persist in circulation to hinder reinfection. To test the possibility that B cell memory, the component of the immune system responsible for antibody production, is expensive to maintain, secondary antibody production against a novel protein [keyhole limpet hemocyanin (KLH)] was compared in food-restricted and ad libitum-fed male deer mice (Peromyscus maniculatus). To determine whether compromised secondary antibody production was solely due to elevated corticosterone independent of resource availability, some food-restricted and ad libitum-fed mice were subjected to unpredictable, chronic (2 h/day) restraint. Mice fed 70% of their ad libitum diet 2 wk after primary antigen challenge produced approximately 95% less IgG against KLH after a second antigen challenge than mice fed ad libitum, even though all mice were fed ad libitum during the secondary antibody response period. Restraint had no effect on secondary IgG production in response to KLH, and corticosterone concentrations 1 day after food restriction did not differ between food-restricted and ad libitum-fed mice. Together, these data imply that secondary antibody responses and the benefits of immunological memory are energetically costly in this species.     

Antigen Experience Shapes Phenotype and Function of Memory Th1 Cells

Primary and secondary (boosted) memory CD8 T cells exhibit differences in gene expression, phenotype and function. The impact of repeated antigen stimulations on memory CD4 T cells is largely unknown. To address this issue, we utilized LCMV and Listeria monocytogenes infection of mice to characterize primary and secondary antigen (Ag)-specific Th1 CD4 T cell responses. Ag-specific primary memory CD4 T cells display a CD62L^loCCR7^hi CD27^hi CD127^hi phenotype and are polyfunctional (most produce IFNγ, TNFα and IL-2). Following homologous prime-boost immunization we observed pathogen-specific differences in the rate of CD62L and CCR7 upregulation on memory CD4 T cells as well as in IL-2+IFNγco-production by secondary effectors. Phenotypic and functional plasticity of memory Th1 cells was observed following heterologous prime-boost immunization, wherein secondary memory CD4 T cells acquired phenotypic and functional characteristics dictated by the boosting agent rather than the primary immunizing agent. Our data also demonstrate that secondary memory Th1 cells accelerated neutralizing Ab formation in response to LCMV infection, suggesting enhanced capacity of this population to provide quality help for antibody production. Collectively these data have important implications for prime-boost vaccination strategies that seek to enhance protective immune responses mediated by Th1 CD4 T cell responses.     

Antigen-independent immune responses after dendritic cell vaccination

The ability of cultured, antigen-loaded dendritic cells (DCs) to induce antigen-specific T cell immunity in vivo has previously been demonstrated and confirmed. Immune monitoring naturally focuses on immunity against vaccine antigens and may thus ignore other effects of DC vaccination. Here we therefore focused on antigen-independent responses induced by DC vaccination of renal cell carcinoma patients. In addition to the anticipated response against the vaccine antigen KLH, vaccination with CD83⁺ monocyte-derived DCs resulted in a strong increase in the ex vivo proliferative and cytokine responses of PBMCs stimulated with LPS or BCG. In addition, LPS strongly enhanced the KLH-induced proliferative and cytokine response of PBMCs. Moreover, proliferative and cytokine responses of PBMCs stimulated with the homeostatic cytokines IL-7 and IL-15 were also clearly enhanced after DC vaccination. In contrast to LPS induced proliferation, which is well known to depend on monocytes, IL-7 induced proliferation was substantially enhanced after monocyte depletion indicating that monocytes limit IL-7 induced lymphocyte expansion. Our data indicate that DC vaccination leads to an increase in the ex vivo responsiveness of patient PBMCs consistent with a DC vaccination induced enhancement of T cell memory. Our findings also suggest that incorporation of bacterial components and homeostatic cytokines into immunotherapy protocols may be useful in order to enhance the efficacy of DC vaccination and that monocytes may limit DC vaccination induced immunity. Supported by a grant to Martin Thurnher from the kompetenzzentrum medizin tirol (kmt), a center of excellence.     

Modulation of mitogen-induced spleen cell proliferation and the antibody-forming cell response by beta-endorphin in vivo

Experiments were conducted which compared the in vivo effects of beta-endorphin (BEP), gamma-endorphin (gamma EP), methionine-enkephalin (Met-ENK), and acetylated BEP(1-27) on the in vitro proliferative response of rat spleen cells to concanavalin A (ConA). In addition, the influence of BEP administration on the primary and secondary antibody-forming cell (AFC) response to the soluble antigen keyhole-limpet hemocyanin (KLH) was examined. Intravenous administration of BEP enhanced the spleen cell proliferative response to ConA assessed 3 hr after a single bolus infusion. Conversely, infusion with AcBEP(1-27) suppressed the proliferative response, whereas no effects of intravenous gamma EP or Met-ENK treatment were observed. The enhancing effect of BEP administration was not detectable 24 hr after a single infusion, but could be maintained over a 44 hr period by multiple infusions. The primary AFC response to KLH was suppressed by a dose of 1 nmole BEP only. On the other hand, the secondary IgG AFC response to KLH was enhanced by 10 pmoles BEP, while the IgM and IgA AFC responses remained unaltered by BEP treatment. The anamnestic in vitro proliferative response of spleen cells cultured with KLH was not altered if BEP was administered at the time of secondary KLH immunization. These results extend previous observations of BEP-induced modulation of in vitro immune function by demonstrating that opioid and nonopioid forms of BEP administered in vivo alter the capacity of spleen cells to proliferate and develop antibody responses to antigen.     

Regulation of antigen-induced lymphoproliferation by BSA gradient-separated T cell fractions

Human T cells from 6 volunteers immunized with keyhole limpet hemocyanin (KLH) or tetanus toxoid were fractionated on bovine serum albumin (BSA) gradients. These T cell fractions were then recombined with autologous unseparated mononuclear cells or unseparated T cells to determine the effect of each fraction on the proliferative (DNA synthetic) response to the immunizing antigen. Using this separation technique, we were able to define 2 fractions of T cells of widely different densities that had a suppressive effect on the proliferative response to KLH of unfractionated T cells. The demonstration of suppressive activity was possible only if cells were tested within 8 wk of immunization with KLH. These suppressive T cell fractions differed not only in density but also in surface characteristics. Fraction 1 cells had a high proportion of Ia+ cells but no Fc receptors for IgG (FcRG-) were seen, whereas cells from fraction 4 (of higher density) were Ia- and were composed of 12.1 +/- 1.2% FcRG+ cells. this suppression was shown to be specific for the immunizing antigen. One individual who had been immunized with KLH for more than 1 yr in which suppression of KLH-induced proliferation was no longer demonstrable was given primary immunization to tetanus toxoid. Suppression could be demonstrated in fractions 1 and 4 specific for tetanus toxoid; there was no demonstrable effect of these T cell fractions on the response to KLH. We postulate that these are different functional populations of suppressor T cells that regulate antigen specific lymphoproliferation.     

Stimulation history dictates memory CD8 T cell phenotype: implications for prime-boost vaccination

Heterologous prime-boost vaccination results in increased frequencies of memory T cells. Although these quantitative effects of reexposure to Ag are well documented, little is known about the impact of boosting on the functional qualities of memory T cells. To address this critical issue, we have used three different types of immunization regimens and examined how boosting effects the function and anatomic location of memory CD8 T cells. We found that memory T cell phenotype differed substantially depending on the number of immunizations and that secondary and tertiary responses resulted in the generation of memory CD8 T cells that retained effector-like properties and showed preferential accumulation in nonlymphoid tissues. These results show that memory differentiation is coupled to the history of Ag experience and that prime-boost vaccination strategies have important consequences on memory CD8 T cell quality and surveillance within mucosal tissues.     

Activation of IL-4 and IL-6 secreting cells by antigen

The number, antigenic specificity and phenotype of cells secreting IL-4 and IL-6 in mice immunized with ovalbumin or keyhole limpet haemocyanin (KLH) in Freund's adjuvant (FA) was studied. The frequency of cells producing either of these cytokines began to rise 6 days post immunization, peaked at 11–14 days post-immunization, and fell to background by 21 days. The number of spleen cells secreting IL-6 was higher than the number producing IL-4 at all time points. Boosting elicited an anamnestic response characterized by a significant increase in the number of cytokine secreting cells within 4 days. Cytokine production was induced in multiple strains of normal mice, and was critically dependent on the use of Complete FA in addition to antigen. Immunization induced IL-4 and IL-6 production in vivo while ‘priming’ additional cells to release these cytokines when reexposed to soluble antigen in vitro. The latter response was antigen specific and was dominated by non-B/non-T cells. Those cells may serve to boost the immune response in cases of persistent or repeated antigenic challenge.     

In vitro immunization to KLH. II. Limiting dilution analysis of antigen-reactive cells in primary and secondary culture

Limiting dilution analysis was used to estimate the frequency of human peripheral blood T lymphocytes that proliferate in response to in vitro immunization with keyhole limpet hemocyanin (KLH). Antigen-reactive cells (ARC) were estimated 9 days after primary immunization with KLH. The ARC frequency of lymphocytes from 12 subjects ranged from 1:23,800 to 1:52,631. Lymphocytes from five of these subjects were also primed for 12 days with KLH, rechallenged in secondary culture with fresh adherent cells and KLH, and assayed 4 days later. The ARC frequency increased to 1:1,123 to 1:7,247, indicating that T cell clones responsive to KLH had expanded during primary culture. In addition, we observed that the proliferative response of lymphocytes from 5 of the 12 subjects were inhibited at high cell concentrations. Depletion of OKT8+ T cells before culturing with KLH however did not alter the inhibitory effect of high concentrations of T cells.     

IL-6 increases primed cell expansion and survival

Cytochrome c-specific CD4 T cells from transgenic donors transferred to syngeneic B10.A mice expand more vigorously upon immunization if exogenous IL-6 is provided during the initial phase of immunization. The resultant increase in the frequency and number of Ag-specific cells is observed in the blood, lymph nodes, spleen, liver, and lung and persists for at least 3 mo. Treatment of immunized recipients with anti-IL-6 or use of IL-6 knockout recipients reduced the frequency of Ag-specific CD4 T cells during a comparable period, indicating that IL-6 is physiologically involved in the expansion of memory and/or effector cells and thus in the persistence of memory. IL-6 did not alter the duration of Ag-presenting activity. Both CFSE dilution studies and labeling with BrdU indicated that IL-6 does not effect proliferative rates of responding CD4 T cells. By contrast, annexin V staining was diminished in responding cells from the IL-6-treated animals, particularly among those cells that had undergone five or more divisions. These results indicate that IL-6 reduces the level of apoptosis among Ag-stimulated cells; thus, it plays a central role in determining numbers of memory and/or effector CD4 T cells in response to immunization over extended periods.     

The ovine nasal mucosa: an alternative tissue site for mucosal immunization

The ovine nasal mucosal environment has histological and ultrastructural features that resemble well-known inductive sites of mucosa-associated lymphoid tissue. In the present study, the nasal mucosa was assessed as a potential mucosal tissue site for delivering vaccines to sheep. Sheep were immunized by either injection with the model antigen, Keyhole Limpet Haemocyanin (KLH), and aluminium hydroxide gel (alum) or by aerosol spray with KLH with and without cholera toxin (CT). Sheep immunized by injection with KLH/alum and aerosol spray with KLH/CT induced strong anti-KLH IgG and IgA serum antibody responses as well as specific T cell memory. Anti-KLH IgG1 responses were significantly higher following immunization by injection and no significant differences in anti-KLH IgG2 responses were detected between groups. Sheep immunized with KLH by aerosol spray without CT did not produce serum antibody and T cell memory responses. Antibody-secreting cells were present in the parotid lymph nodes (draining lymph nodes) of sheep immunized with KLH/alum and KLH/CT, but secreted only Ag-specific IgG1, and not IgG2 or IgA. These results suggest that aerosolization of soluble antigen formulations with CT may provide an alternative method of delivering nasal vaccines to sheep and other large animal species, and that further improvements in antigen penetration of nasal tissues may dramatically improve the strength of the immune response.     

Vaccination alone or in combination with pyridostigmine promotes and prolongs activation of stress-activated kinases induced by stress in the mouse brain

Gulf war illnesses (GWI) are currently affecting thousands of veterans. To date, the molecular mechanisms underlying the pathogenesis of these illnesses remain unknown. During Gulf war I, military personnel were exposed to multiple stressors, one or more vaccines, pyridostigmine (PY), and other chemicals. In our previous studies, we found that stress induces activation of mitogen activated protein-kinase kinase 4 (MKK4) and c-Jun-N-terminal kinase (JNK) in the mouse brain (Liu et al. 2004). Our working hypothesis is that stress, vaccination, and PY may synergistically induce activation of MKK4 and JNK in the brain, leading to over-activation of these kinases and neurological injuries. To test our hypothesis, we examined the effect of keyhole limpet hemocyanin (KLH) immunization alone or in combination with PY on activation of MKK4 and JNK induced by stress. We found that KLH immunization alone had a small effect on MKK4 or JNK activity but it significantly enhanced and prolonged activation of these kinases induced by stress, from a few hours to several days. Additionally, KLH immunization caused activation of p38MAPK. PY treatment further enhanced and prolonged activation of these kinases induced by stress in combination with KLH immunization and triggered activation of caspase-3. Our current studies suggest that stress, vaccination, and PY may synergistically act on multiple stress-activated kinases in the brain to cause neurological impairments in GWI.     

Immune responses following neonatal DNA vaccination are long-lived, abundant, and qualitatively similar to those induced by conventional immunization

Virus infections are devastating to neonates, and the induction of active antiviral immunity in this age group is an important goal. Here, we show that a single neonatal DNA vaccination induces cellular and humoral immune responses which are maintained for a significant part of the animal's life span. We employ a sensitive technique which permits the first demonstration and quantitation, directly ex vivo, of virus-specific CD8(+) T cells induced by DNA immunization. One year postvaccination, antigen-specific CD8(+) T cells were readily detectable and constituted 0.5 to 1% of all CD8(+) T cells. By several criteria-including cytokine production, perforin content, development of lytic ability, and protective capacity-DNA vaccine-induced CD8(+) memory T cells were indistinguishable from memory cells induced by immunization with a conventional (live-virus) vaccine. Analyses of long-term humoral immune responses revealed that, in contrast to the strong immunoglobulin G2a (IgG2a) skewing of the humoral response seen after conventional vaccination, IgG1 and IgG2a levels were similar in DNA-vaccinated neonatal and adult animals, indicating a balanced T helper response. Collectively, these results show that a single DNA vaccination within hours or days of birth can induce long-lasting CD8(+) T- and B-cell responses; there is no need for secondary immunization (boosting). Furthermore, the observed immune responses induced in neonates and in adults are indistinguishable by several criteria, including protection against virus challenge.     

The in vivo elimination of CD4+ T cells prevents the induction but not the expression of carrier-induced epitopic suppression.

Injection of mice with an immunogenic dose of carrier (keyhole limpet hemocyanin (KLH)) followed by immunization with hapten-carrier conjugate (TNP-KLH) selectively suppresses anti-hapten antibody response. In this study, the cellular basis of this epitopic suppression and also of the suppression induced by a high dose of carrier were analyzed by in vivo depletion of CD4+ or CD8+ T cell subsets by using mAb. The mAb treatments were performed either at the time of carrier priming or at the time of hapten-carrier immunization. The elimination of CD8+ T cells has not modified the anti-carrier antibody response, whether this treatment was performed at the time of KLH-priming or during TNP-KLH immunization. Moreover, the in vivo treatment with the anti-CD8 mAb did not modify the carrier-induced epitopic suppression induced either by a low immunogenic dose of KLH or by a high dose of this Ag. The elimination of CD4+ T cells at the time of KLH immunization has prevented the induction of a memory response to KLH, clearly establishing that CD4+ T cells are essential in memory B cell development to T-dependent Ag. Moreover, this treatment has totally abrogated the epitopic suppression induced either by low or high dosages of KLH. In contrast, the in vivo elimination of CD4+ T cells after carrier immunization did not abolish the secondary anti-carrier antibody response and did not prevent the expression of epitopic suppression. These data indicate that primed CD4+ T cells are required neither for memory B cell expression nor for the expression of suppression. Finally, once induced, the suppression can be evidenced after in vivo depletion of both primed CD4+ and CD8+ T cells. These data support the view that epitopic suppression is induced through the expansion of carrier-specific B cells and resulted from intramolecular antigenic competition between hapten and carrier epitopes.     

壳聚糖盐酸盐稳定乳液的制备及免疫效果分析

为了解决油乳佐剂在诱导细胞免疫方面的不足,引入正电荷的壳聚糖盐以稳定乳液,从而提升细胞免疫应答。本研究选取卵清蛋白为模式抗原,分别制备了单抗原疫苗、商品佐剂疫苗和乳液疫苗,表征了乳液的粒径、电势及抗原吸附率等参数。通过肌肉注射免疫BALB/c小鼠,检测免疫后抗体和细胞因子的分泌水平、淋巴细胞的活化水平以评价乳液的免疫效果。结果显示壳聚糖盐酸盐可有效稳定乳液,其粒径在600 nm左右,荷正电并对抗原吸附率达90%以上。免疫动物后,该乳液可有效提升血清特异性抗体水平并增强IL-4的分泌,显著提高抗原特异性CD8⁺T细胞的比例,提升细胞毒性T淋巴细胞(cytotoxic T lymphocyte, CTL)表面活化分子的表达水平,诱导高效的细胞免疫应答。此外,乳液能够有效提高记忆T细胞(CD44⁺CD62L⁺)的水平。综上所述,以壳聚糖盐酸盐稳定的乳液为佐剂能有效提升体液及细胞免疫效果,并有望起长期保护作用。     

Depletion of CD4+ T cells during immunization with nonviable Listeria monocytogenes causes enhanced CD8+ T cell-mediated protection against listeriosis

Immunization of mice with nonviable Listeria monocytogenes generates an insufficient CD8(+) T cell response and consequently only limited protection against subsequent L. monocytogenes infection. We have recently demonstrated that depletion of regulatory CD4(+) T cells during immunization significantly enhances CD8(+) T cell responses. In the present study, we determined the impact of CD4(+) T cell depletion on the CD8(+) T cell response against heat-killed LISTERIA: Treatment of mice with anti-CD4 mAb during boost immunization with heat-killed Listeria significantly increased numbers of Listeria-specific CD8(+) T cells and improved protection against subsequent infection with L. monocytogenes. During challenge infection, numbers of Listeria-specific CD8(+) T cells were enhanced, and these cells expressed effector functions in terms of IFN-gamma production. In summary, we demonstrate that combining nonviable L. monocytogenes vaccination and CD4(+) T cell depletion improves generation of long-lasting and functional Listeria-specific CD8(+) memory T cells.     

Transition in the Character of Immunological Memory in Mice after Immunization

The immunological memory in antibody response of mice to bovine serum albumin was investigated at the level of IgM and IgG antibody-forming cells. The antigen at a dose much lower than required for eliciting a detectable level of the primary antibody response could latently activate the immune machinery to an extent adequate for specific recall, whereas higher doses of antigen were effective in evoking strong anamnestic response. The potentiality to develop the anamnestic response was found even in the latent phase of the primary antibody response and was maintained for more than 2 months. The immunological memory acquired in an early phase after the primary immunization mainly involved IgM antibody response and late memory concerned IgG response.     

IL-21 receptor is critical for the development of memory B cell responses

Development of long-term humoral immunity, characterized by the formation of long-lived plasma cells (PCs) in the bone marrow and memory B cells, is a critical component of protective immunity to pathogens, and as such it is the major goal of vaccination. However, the mechanisms involved in the generation of long-term humoral immunity remain poorly understood. In this study, we used IL-21R-deficient (IL-21R.KO) mice to examine the role of the IL-21 pathway in the development of the B cell memory response. Primary IgG serum Ab responses to the T cell-dependent Ag 4-hydroxy-3-nitrophenylacetyl (NP) hapten conjugated to chicken γ globulin were delayed in IL-21R.KO mice, but reached normal titers within 3 to 4 wk of immunization. IL-21R.KO mice formed germinal centers and generated normal numbers of PCs in their bone marrow. Additionally, memory B cell formation was similar in wild-type and IL-21R.KO mice. However, NP-specific memory B cells and PCs failed to expand following secondary immunization of IL-21R.KO mice, and consequently, secondary IgG Ab responses to NP hapten conjugated to chicken γ globulin were significantly impaired. These results identify the IL-21 pathway as a critical component of the memory B cell response.     

CD4+ CD25+ T cells regulate vaccine-generated primary and memory CD8+ T-cell responses against herpes simplex virus type 1

It has become evident that naturally occurring CD25(+) regulatory T cells (T(reg) cells) not only influence self-antigen specific immune response but also dampen foreign antigen specific immunity. This report extends our previous findings by demonstrating that immunity to certain herpes simplex virus (HSV) vaccines is significantly elevated and more effective if T(reg) cell response is curtailed during either primary or recall immunization. The data presented here show that removal of CD25(+) T(reg) cells prior to SSIEFARL-CpG or gB-DNA immunization significantly enhanced the resultant CD8(+) T-cell response to the immunodominant SSIEFARL peptide. The enhanced CD8(+) T-cell reactivity in T(reg) cell-depleted animals was between two- and threefold and evident in both acute and memory stages. Interestingly, removal of CD25(+) T(reg) cells during the memory recall response to plasmid immunization resulted in a twofold increase in CD8(+) T-cell memory pool. Moreover, in the challenge experiments, memory CD8(+) T cells generated with plasmid DNA in the absence of T(reg) cells cleared the virus more effectively compared with control groups. We conclude that CD25(+) T(reg) cells quantitatively as well as qualitatively affect the memory CD8(+) T-cell response generated by gB-DNA vaccination against HSV. However, it remains to be seen if all types of vaccines against HSV are similarly affected by CD25(+) T(reg) cells and if it is possible to devise means of limiting T(reg) cell activity to enhance vaccine efficacy.     

Rapid development of T cell memory

Prime-boost immunization is a promising strategy for inducing and amplifying pathogen- or tumor-specific memory CD8 T cell responses. Although expansion of CD8 T cell populations following the second Ag dose is integral to the prime-boost strategy, it remains unclear when, after priming, memory T cells become competent to proliferate. In this study, we show that Ag-specific CD8 T cells with the capacity to undergo extensive expansion are already present at the peak of the primary immune response in mice. These early memory T cells represent a small fraction of the primary immune response and, at early time points, their potential to proliferate is obscured by large effector T cell populations that rapidly clear Ag upon reimmunization. With sufficient Ag boosting, however, secondary expansion of these memory cells can be induced as early as 5-7 days following primary immunization. Importantly, both early and delayed boosting result in similar levels of protective immunity to subsequent pathogen challenge. Early commitment and differentiation of memory T cells during primary immunization suggest that a short duration between priming and boosting is feasible, providing potential logistic advantages for large-scale prime-boost vaccination of human populations.