Th1-type immune response to a Coccidioides immitis antigen delivered by an attenuated strain of the non-invasive enteropathogen Vibrio cholerae

The antigen-2 or proline rich antigen (Ag2/PRA) from Coccidioides immitis, known to protect mice against experimental Coccidioidomycosis, was expressed in the genetically attenuated cholera vaccine candidate Vibrio cholerae 638 and its thymine auxotrophic derivative 638T. Intranasal immunization of mice with strains producing Ag2/PRA induced serum vibriocidal antibody and Ag2/PRA-specific total IgG responses in outbred Swiss Webster and inbred BALB/c mice. Analysis of IgG subclasses showed a predominance of IgG2a subclass antibodies. Lymphocytes from immunized mice stimulated with pure Ag2/PRA showed a significant proliferative response with production of interferon-gamma. Positive selection for plasmid maintenance in vivo did not enhance immune response to Ag2/PRA. These results demonstrate that genetically attenuated strains of the non-invasive pathogen V. cholerae can be used to express and deliver foreign antigens to stimulate a Th1 type of immune response.     

Breaking T cell tolerance with foreign and self co-immunogens. A study of autoimmune B and T cell epitopes of cytochrome c.

The initiation of autoimmune B cell and T cell responses by self Ag or by foreign pathogens (molecular mimics) is not well understood. In the present study, cytochrome c (cyt c) was used as a model autoantigen to investigate how self-proteins are involved in the priming of autoimmune T cell responses. Immunization with foreign cyt c has been extensively analyzed in previous studies as a model for both humoral and cellular immune responses. Mice do not, however, make antibody or T cell responses to immunization with self (mouse) cyt c. In addition, T cell tolerance can be broken by autoreactive B cells that are readily elicited by immunization with cross-reactive foreign cyt c. These immune B cells presumably bind self cyt c and process and present the self Ag to stimulate an autoreactive T cell response. Autoreactive T cell clones derived by this mechanism are all specific for determinants within amino acids 1-80 of the cyt c protein presented by I-Ek. No T cell responses were observed to the carboxyl terminal 81-104 fragment that dominates the response to foreign cyt c. All clones derived in this study are stimulated by a polypeptide encompassing amino acids 54-68 and utilized the V beta 8.2 TCR gene. In contrast, T cells stimulated by foreign cyt c did indeed respond to fragment 81-104 and appear to utilize alternate TCR genes. Our data demonstrate that B cells specific for linear determinants distributed along the entire length of the foreign cyt c molecule can provide the stimulus required for breaking T cell tolerance to self cyt c. The applications of this work to understanding the mechanisms of autoimmune disease are discussed.     

The serotonergic somatosensory projection to the tectum of normal and eyeless salamanders

The spinotectal somatosensory projection was compared in normal, genetically eyeless, and embryonically manipulated salamanders. In normal animals, serotonin fluorescence was restricted to the intermediate tectalneuropil. This same region showed both high levels of serotonin uptake and somatosensory single unit electrical activity. In mutant eyeless salamanders and in normal animals enucleated early in development, serotonin fluorescence, serotonin uptake, and somatosensory activity were present in the superficial tectal neuropil. One-eyed animals, either genetically normal axolotls with one eye enucleated embryonically or genetically eyeless animals in which a normal eye had been transplanted, showed normal intermediate serotonin fluroescence and somatosensory physiology in the visually innervated half-tectum. In the visually uninnervated half-tectum, they showed superficial serotonin fluorescence and somatosensory physiology. In normal animals, 5,7-dihydroxytryptamine (5,7-DHT), a specific poison for serotonergic fibers, eliminated physiological responses in the contralateral somatosensory tectal region. The 5,7-DHT poisoning also abolished U.V.-induced serotonin fluorescence in the intermediate tectal neuropil. These results are discussed in terms of (1) evidence for serotonin as a central neurotransmitter for somatosensory information in the tectum, (2) the effects of eyelessness on tectal organization, and (3) related results in other animals.     

The cellular location of a foreign B cell epitope expressed by recombinant bacteria determines its T cell-independent or T cell-dependent characteristics.

We have targeted two foreign B cell antigenic determinants to different locations in the Escherichia coli cell to examine what effect this had on antibody responses elicited by the recombinant bacteria. The two epitopes were the 132-145 peptide from the PreS2 region of hepatitis B virus and the C3 neutralization epitope of poliovirus type 1. They were each expressed in two forms either on the surface, as part of the outer-membrane protein LamB, or soluble in the periplasm, as part of the periplasmic protein MalE. When live bacteria expressing the foreign epitope at the cell surface were used for immunization of mice, they induced T cell-independent antibody responses characterized by a rapid induction of IgM and IgG antibodies. In contrast, when the same foreign epitope was inserted into the MalE protein, the antibody response was only detectable after 3 wk, belonged only to the IgG class and was strictly T cell dependent. This study has therefore identified two major pathways by which epitopes expressed by bacterial cells can stimulate specific antibody responses. The first pathway is mediated by direct activation of B cells by bacterial cell-surface Ag and does not require T cell help. The second pathway is T cell dependent and concerns Ag that can be released from the bacteria in a soluble form. We have also studied the effect of the exact position of the B cell antigenic determinant within the LamB protein and with respect to the outer membrane by comparing the immunogenicity of the PreS epitope inserted at three different permissive sites of LamB. The data indicated that to obtain an antibody response with intact bacteria, the epitope must be protruding sufficiently from the outside of the outer membrane. In contrast, when semipurified hybrid proteins were used as immunogen, the exact position of the B cell antigenic determinant within solubilized LamB protein does not influence its immunogenicity.     

Absence of functional inducible NO synthase enhances the efficacy of tolerance induced by high dose antigen feeding

Recent studies have suggested that IL-12 and IFN-gamma may impair the ability of fed Ag to induce systemic tolerance. Because both of these cytokines can function to directly or indirectly induce inducible NO synthase (iNOS) expression, we have investigated whether the functional expression of iNOS regulates oral tolerance. C57BL/6J wild-type or C57BL/6J NOS2(-/-) mice were gavaged with a single dose of 20 mg of keyhole limpet hemocyanin (KLH), followed by s.c. immunization with KLH/CFA. In the absence of feeding Ag, several parameters of the immune response were more robust in C57BL/6J NOS2(-/-) mice following KLH/CFA immunization, including the magnitude of the delayed-type hypersensitivity response, the proliferative response, and the production of IFN-gamma and IL-2 by Ag-activated draining lymph node cells. These heightened responses in the C57BL/6J NOS2(-/-) mice are still effectively inhibited by feeding KLH. Feeding KLH to the C57BL/6J NOS2(-/-) mice elicited heightened TGF-ss1 production by Ag-activated lymphocytes, as well as augmented total IgG, IgG1, and IgG2a responses to KLH/CFA compared with that seen in Ag-fed wild-type mice. Feeding Ag to the NOS2(-/-) mice suppressed proliferative responses and IFN-gamma production, while increasing IL-4 production and the IgG1/IgG2a ratio even following a booster immunization of KLH/CFA. Administrating L-N:(6)-(1-iminoethyl)-lysine. 2HCl to wild-type mice during the period of Ag feeding reproduced the high TGF-ss1 production seen in Ag-activated lymphocytes from Ag-fed NOS2(-/-) mice. Feeding KLH is followed by transient up-regulation of NOS2 mRNA expression in the Peyer's patches of wild-type mice. Selective inhibition of NOS2 may be a simple way to augment tolerogenic mucosal immune responses.     

Anti-peptide antibody production elicited by in vitro immunization of human peripheral blood mononuclear cells

Human monoclonal antibodies have great potential for use in the treatment of various diseases. We have established an in vitro immunization protocol for inducing antigen-specific antibody production from human peripheral blood mononuclear cells (PBMCs). In the in vitro immunization protocol, PBMCs are pretreated with L-leucyl-L-leucine methyl ester (LLME) to remove suppressive cells, and are sensitized and cultured with a soluble antigen in the presence of IL-2, IL-4 and muramyl dipeptide for 8 d, and then an antigen-specific antibody is produced. In this study, we examined the novel possibility of an in vitro immunization protocol, specifically, whether LLME-treated PBMCs can be sensitized with a peptide antigen to produce an anti-peptide antibody. The results indicate that antigen-specific immune responses were elicited by a peptide antigen derived from rice allergen, a cholera toxin B subunit, and TNF-alpha as a sensitizing antigen in in vitro immunization. These results suggest that the in vitro immunization protocol is applicable in the generation of an anti-peptide antibody against various antigens, including food allergens, foreign antigens, and self-antigens.     

Role of B7 in T cell tolerance

The induction of effective immune responses requires costimulation by B7 molecules, and Ag recognition without B7 is thought to result in no response or tolerance. We compared T cell responses in vivo to the same Ag presented either by mature dendritic cells (DCs) or as self, in the presence or absence of B7. We show that Ag presentation by mature B7-1/2-deficient DCs fails to elicit an effector T cell response but does not induce tolerance. In contrast, using a newly developed adoptive transfer system, we show that naive OVA-specific DO11 CD4+ T cells become anergic upon encounter with a soluble form of OVA, in the presence or absence of B7. However, tolerance in DO11 cells transferred into soluble OVA transgenic recipients can be broken by immunization with Ag-pulsed DCs only in B7-deficient mice and not in wild-type mice, suggesting a role of B7 in maintaining tolerance in the presence of strong immunogenic signals. Comparing two double-transgenic models--expressing either a soluble or a tissue Ag--we further show that B7 is not only essential for the active induction of regulatory T cells in the thymus, but also for their maintenance in the periphery. Thus, the obligatory role of B7 molecules paradoxically is to promote effective T cell priming and contain effector responses when self-Ags are presented as foreign.     

Recombinant human IL-2 overcomes genetic nonresponsiveness to malaria sporozoite peptides. Correlation of effect with biologic activity of IL-2

Current malaria vaccine strategies focus on subunit vaccines that contain one or a limited number of malaria Ag. However, there is widespread nonresponsiveness to many of these Ag probably resulting from Ir gene control. Using a congenic mouse model, we demonstrated that human rIL-2 (as an adjuvant) can overcome Ir gene controlled low immune responsiveness to peptide malaria Ag vaccine candidates [R32tet32, R32LR, and Th2R-NP (NANP)5NA] as determined by the antibody response, providing it is emulsified with the Ag during immunization. This effect is not caused by IL-2 merely acting as a foreign protein and stimulating noncognate help; it requires biologic activity of the IL-2, as determined by studying the effect of inactive rIL-2, which has minimal biologic activity but which has retained its antigenicity. IL-2 does not appear to be working by an effect on priming of specific Th, and IL-2 cannot overcome an Ir gene controlled low T cell proliferative response. IL-2 may have a role to play in human vaccine development where a high titer antibody response to a subunit vaccine is required.     

Mechanisms of peripheral tolerance following intracameral inoculation are independent of IL-13 or STAT6

The peripheral tolerance that is elicited by the anterior chamber-associated immune deviation (ACAID) protocol is characterized by impairment of Th1 responses such as delayed-type hypersensitivity. It has been proposed that suppression of Th1 responses is mediated by a deviation toward Th2 responses. Because NKT cells have a prominent role in ACAID and NKT cell-derived IL-13 is required in a tumor model of tolerance, we postulated that NKT cell-derived Th2 cytokines might have a role in ACAID. However, contrary to the tumor model, in this study we show that NKT cells from IL-13-deficient mice or IL-4/IL-13 double deficient mice were able to reconstitute the capability of J alpha18-deficient mice (lacking invariant NKT) to develop peripheral tolerance postintracameral inoculation of Ag. Also, we were able to induce peripheral tolerance directly in IL-13-deficient, IL-4/IL-13-double deficient, and STAT6-deficient mice by inoculation of Ag into their eye. We conclude that neither IL-4 nor IL-13 cytokines are required for the generation of efferent CD8+ T regulatory cells during eye-induced peripheral tolerance. We propose that Ags inoculated into the anterior chamber of the eye induce the immunoresponse to deviate from producing immune T effector cells to producing efferent T regulatory cells, rather than deviating from Th1- to Th2-type effector cells.     

Dendritic cell-based xenoantigen vaccination for prostate cancer immunotherapy.

Many tumor-associated Ags represent tissue differentiation Ags that are poorly immunogenic. Their weak immunogenicity may be due to immune tolerance to self-Ags. Prostatic acid phosphatase (PAP) is just such an Ag that is expressed by both normal and malignant prostate tissue. We have previously demonstrated that PAP can be immunogenic in a rodent model. However, generation of prostate-specific autoimmunity was seen only when a xenogeneic homolog of PAP was used as the immunogen. To explore the potential role of xenoantigen immunization in cancer patients, we performed a phase I clinical trial using dendritic cells pulsed with recombinant mouse PAP as a tumor vaccine. Twenty-one patients with metastatic prostate cancer received two monthly vaccinations of xenoantigen-loaded dendritic cells with minimal treatment-associated side effects. All patients developed T cell immunity to mouse PAP following immunization. Eleven of the 21 patients also developed T cell proliferative responses to the homologous self-Ag. These responses were associated with Ag-specific IFN-gamma and/or TNF-alpha secretion, but not IL-4, consistent with induction of Th1 immunity. Finally, 6 of 21 patients had clinical stabilization of their previously progressing prostate cancer. All six of these patients developed T cell immunity to human PAP following vaccination. These results demonstrate that xenoantigen immunization can break tolerance to a self-Ag in humans, resulting in a clinically significant antitumor effect.     

Characterization of streptococcal antigen-specific CD8+, MHC class I-restricted, T cell clones that down-regulate in vitro antibody synthesis.

T cell clones were generated from the peripheral blood of rhesus monkeys that had been immunized with a soluble Mr 185,000 Ag (SAI/II) derived from Streptococcus mutans. The clones were CD3+ CD8+ CD4- alpha beta TCR+ and were specifically stimulated to proliferate by SAI/II. The proliferative responses of the cloned cells were class I restricted, as demonstrated by reconstitution of the cloned T cells with APC matched at various MHC class I and II loci, as well as by inhibition with anti-class I and not anti-class II mAb. The function of the CD8+ cloned cells was examined in vitro for their effect on antibody synthesis by Ag-stimulated CD4+ cells and B cells from immunized animals. Indeed, four of the five clones suppressed SAI/II-specific IgG antibody synthesis when activated with SAI/II and the appropriate MHC-matched APC. Although activation of the suppressor clones was Ag specific, the effector function of the suppression of antibody synthesis was Ag nonspecific. The latter was probably mediated by lymphokines and, indeed, the culture supernatant generated by stimulating the cloned CD8+ cells with anti-CD3 mAb suppressed both the specific and nonspecific antibody synthesis. Cytotoxicity studies showed that all five CD8+ clones showed a low level of lectin-dependent cytotoxicity. However, because four of the five clones expressed significant suppression of antibody synthesis, the suppressor activity was unlikely to be a function of the weak cytotoxicity. The results suggest that immunization of rhesus monkeys with a soluble streptococcal Ag induced CD8+ alpha beta TCR+ T cell clones that show SAI/II-specific, MHC class I-restricted proliferative responses and nonspecific down-regulatory function of in vitro antibody synthesis.     

Development of optokinetic neuronal responses in the pretectum and horizontal optokinetic nystagmus in unilaterally enucleated rats

Responses of single units to constant-velocity rotations of the visual surround (0.25-10 degrees/s) were studied in the pretectum of unilateral enucleated rats at different ages. Enucleation was performed either in the first postnatal week ("early" enucleated rats) or in the adult stage ("late" enucleated rats). Pretectal unitary responses were recorded in early enucleated animals at postnatal day 20-21, 36-49 and, in both experimental groups, in the adult stage. Optokinetic ocular nystagmus was studied in early and late enucleated rats in the adult stage. Gain of optokinetic nystagmus in temporo-nasal stimulus direction was not changed for visual surround rotations of up to 20 degrees/s compared to controls in monocular viewing conditions. At higher stimulus velocities, however, the gain dropped. In naso-temporal stimulus direction, optokinetic nystagmus was improved in gain for optokinetic pattern motions of up to 5-10 degrees/s. There were only minor differences in the gain behaviour of optokinetic nystagmus obtained from early or late enucleated rats. The optokinetic responses of pretectal neurons obtained from early and late enucleated rats were reduced in sensitivity by more than 50%. The response patterns of neurons recorded in the contralateral pretectum relative to the intact eye were shifted by a large amount from directional selective to directional nonselective response types. No such changes were obtained in the ipsilateral pretectum. In contrast to normal rats, there were very few directional selective units responding to temporo-nasal pattern motion. On the other hand, a large proportion of directional selective units responded to naso-temporal pattern motion. These latter units were found in both early and late enucleated rats. A similar response type has previously been described for intact young rats but not for adult rats. The velocity tuning curve of pretectal units studied in the adult stage was similar in shape in early and late enucleated rats and resembled that obtained from enucleated or intact young animals. Our results show that response sensitivity, direction and velocity tuning of pretectal units depend crucially on retinal afferent input originating from both eyes. The data suggest that the response characteristics of many of the pretectal units that are considered to be important for mediating optokinetic reflexes depend on interpretectal signal processing using commissural connections. There is very little evidence for an adaptative structural plasticity of the optokinetic system following loss of one eye. The reduced asymmetry observed in gain of optokinetic responses correlated in both early and late enucleated rats with the shifts observed in the distribution of pretectal unitary response patterns.     

Systemic immunological tolerance to ocular antigens is mediated by TRAIL-expressing CD8+ T cells

Systemic immunological tolerance to Ag encountered in the eye restricts the formation of potentially damaging immune responses that would otherwise be initiated at other anatomical locations. We previously demonstrated that tolerance to Ag administered via the anterior chamber (AC) of the eye required Fas ligand-mediated apoptotic death of inflammatory cells that enter the eye in response to the antigenic challenge. Moreover, the systemic tolerance induced after AC injection of Ag was mediated by CD8(+) regulatory T cells. This study examined the mechanism by which these CD8(+) regulatory T cells mediate tolerance after AC injection of Ag. AC injection of Ag did not prime CD4(+) T cells and led to increased TRAIL expression by splenic CD8(+) T cells. Unlike wild-type mice, Trail(-/-) or Dr5(-/-) mice did not develop tolerance to Ag injected into the eye, even though responding lymphocytes underwent apoptosis in the AC of the eyes of these mice. CD8(+) T cells from Trail(-/-) mice that were first injected via the AC with Ag were unable to transfer tolerance to naive recipient wild-type mice, but CD8(+) T cells from AC-injected wild-type or Dr5(-/-) mice could transfer tolerance. Importantly, the transferred wild-type (Trail(+/+)) CD8(+) T cells were also able to decrease the number of infiltrating inflammatory cells into the eye; however, Trail(-/-) CD8(+) T cells were unable to limit the inflammatory cell ingress. Together, our data suggest that "helpless" CD8(+) regulatory T cells generated after AC injection of Ag enforce systemic tolerance in a TRAIL-dependent manner to inhibit inflammation in the eye.     

IL-1 is an effective adjuvant for mucosal and systemic immune responses when coadministered with protein immunogens

Mucosal immunization with soluble protein Ag alone may induce Ag-specific tolerance, whereas mucosal immunization with Ag in the presence of a mucosal adjuvant may induce Ag-specific systemic and mucosal humoral and cell-mediated immune responses. The most widely used and studied mucosal adjuvant is cholera toxin (CT). Although the mechanism of adjuvanticity of CT is not completely understood, it is known that CT induces mucosal epithelial cells to produce the proinflammatory cytokines IL-1, IL-6, and IL-8 and up-regulates macrophage production of IL-1 and the costimulatory molecule B7.2. Because IL-1 may duplicate many of the activities of CT, we evaluated IL-1alpha and IL-1beta for their ability to serve as mucosal adjuvants when intranasally administered with soluble protein Ags. IL-1alpha and IL-1beta were as effective as CT for the induction of Ag-specific serum IgG, vaginal IgG and IgA, systemic delayed-type hypersensitivity, and lymphocyte proliferative responses when intranasally administered with soluble protein Ag. Our results indicate that IL-1alpha and IL-1beta may be useful as mucosal vaccine adjuvants. Such an adjuvant may be useful, and possibly required, for vaccine-mediated protection against pathogens that infect via the mucosal surfaces of the host such as HIV.     

Eye-specific segregation of optic afferents in mammals,fish, and frogs: The role of activity

Eye-specific patches or stripes normally develop in the visual cortex and superior colliculus of many (but not all) mammals and are also formed, after surgically produced binocular innervation, in the optic tectum of fish and frogs. The segregation of ocular dominance patches or columns has been studied using a variety of anatomical pathway-tracing techniques, by electrophysiological recording of postsynaptic units or field potentials, and by the 2-deoxyglucose method following visual stimulation of only one eye. In the tectum of both fish and frogs and in the cortex and colliculus of mammals, eye-specific patches develop from initially diffuse, overlapping projections. Of the various mechanisms that might cause such segregation, the evidence favors an activity-dependent process that stabilizes synapses from the same eye because of their correlated activity. First, several environmental manipulations affect the segregation of afferents in visual cortex: strabismus and alternate monocular exposure apparently enhance segregation, whereas dark rearing slows the segregation process, and monocular deprivation causes the experienced eye to form larger patches at the expense of those of the deprived eye. Second, blocking activity in both eyes is effective in preventing the segregation both in the tectum of fish and frog and in the visual cortex of cat. With the eyes blocked, alternate stimulation of the optic nerves permits the segregation of ocular dominance, at least onto single cells in the cat visual cortex. These findings are discussed in terms of an activity-dependent stabilization of those synapses having correlated activity (those from neighboring ganglion cells within one eye) but not of those lacking correlated activity (those from left and right eyes). We suggest that the eye-specific patches represent a compromise between total segregation of the projections from the two eyes and the formation of a single continuous retinotopic map across the surface of the cortex or tectum.     

Immunogenicity of synthetic peptides derived from the sequences of a Streptococcus mutans cell surface antigen in nonhuman primates

The immunogenicity and antigenicity of synthetic peptides (SP) derived from the sequences of a cell surface Ag of Streptococcus mutans were investigated in macaque monkeys. Immunization with the free peptides of 11, 17, and 21 residues failed to elicit serum antibodies or T cell responses. However, immunization with the SP17 and SP21 linked to tetanus toxoid (TT) as a carrier elicited serum antibodies and proliferative responses of lymphocytes, not only to the SP but also to the native streptococcal Ag. In vivo recall of SP-TT immunized monkeys with suboptimal doses of the native streptococcal Ag resulted in a significant increase in antibodies, both to the SP and the streptococcal Ag, confirming that the SP shares antigenic epitopes with the native Ag. B and T cell epitopes were then determined and a B cell epitope was found in residues 8-13, whereas an overlapping T cell epitope was located in residues 7-15. The T cell epitope has an amino-terminal leucine and carboxy-terminal glycine and alanine added to residues 8-13 of the B cell epitope. In spite of the B and T cell epitopes being expressed in SP17 (residues 1-15), the monomer failed to induce serum antibodies without a carrier. However, immunization with a dimer of SP17 elicited both serum antibodies and proliferative responses of lymphocytes without a carrier. The results suggest that the monomeric SP17 is not immunogenic and needs to be dimerised in order to elicit antibodies and T cell responses, both to the SP and to the streptococcal Ag.     

Regulatory CD8+ T cells control neonatal tolerance to a Th2-mediated autoimmunity

Exposure of newborn animals to a foreign Ag may result in immunological tolerance to that specific Ag, a phenomenon called neonatal tolerance. We have previously reported that neonatal administration to Brown-Norway rats of mercury, a heavy metal toxicant, induces a dominant tolerance, specific for the chemical otherwise responsible for Th2 cell-mediated autoimmune responses in this susceptible strain of rats. Neonatal exposure to Ags can prime immunity, rather than inactivate or delete responses, and sustain regulatory functions effective against autoreactive T cells. Here, we address whether such a tolerant response is due to the generation of regulatory cells. The results suggest that the CD8(+) T cell subset is involved in neonatal tolerance to mercuric salt-induced Th2 autoimmune disease. Thus, we demonstrate that in vivo CD8 depletion breaks tolerance following mercury recall in animals under a neonatal tolerance protocol. Furthermore, adoptive cotransfer of splenocytes from naive and tolerant rats as well as transfer of CD8(+) T cells from tolerant animals prevent naive syngeneic rats from developing pathologic Th2 immune responses. These observations indicate that CD8(+) T cells are endowed with regulatory functions in neonatal tolerance and mediate active suppression. Moreover, neonatal tolerance induced the expansion of CD8(+)CD45RC(high) T cells and the emergence of a high percentage of IFN-gamma-synthesizing CD8(+) T cells, which probably reflects the implication of regulatory Tc1 cells. Thus, in vivo induction of neonatal tolerance suppresses Th2 autoimmune responses via generation of a CD8(+) cell-mediated regulatory response.     

Human dendritic cells transfected with RNA encoding prostate-specific antigen stimulate prostate-specific CTL responses in vitro

Although immunological tolerance to self Ags represents an important mechanism to prevent normal tissue injury, there is growing evidence that tolerance to tumor Ags, which often represent normal peripherally expressed proteins, is not absolute and can be effectively reverted. Prostate-specific Ag (PSA) is a self Ag expressed by both normal and malignant prostatic epithelium, and therefore offers a unique opportunity to examine the ability of self Ags to serve as specific CTL targets. In this study, we investigated the efficacy of autologous dendritic cells (DC) transfected with mRNA encoding PSA to stimulate CTL against PSA Ags in vitro. Ag in form of RNA carries the advantage to encode multiple epitopes for many HLA alleles, thus permitting induction of CTL responses among many cancer patients independent of their HLA repertoire. In this study, we show that PSA mRNA-transfected DC were capable of stimulating primary CTL responses against PSA Ags in vitro. The PSA-specific CTL did not cross-react with kallikrein Ags, a protein, which shares significant homology with PSA, suggesting that harmful autoimmune toxicity may not represent a significant problem with this approach. PSA RNA-transfected DC generated from male or female healthy volunteers or from cancer patients were equally effective in stimulating PSA-specific CTL in vitro, implying that neither natural tolerance to PSA Ags nor tumor-mediated T cell anergy may represent major barriers for CTL generation against the self Ag PSA. This study provides a preclinical rationale for using PSA RNA-transfected DC in active or adoptive immunization protocols.     

T- and B-cell nonresponsiveness to self-alphaB-crystallin in SJL mice prevents the induction of experimental allergic encephalomyelitis

The myelin-associated stress protein alphaB-crystallin triggers strong proliferative responses and IFN-gamma production by human T cells and it is considered a candidate autoantigen in multiple sclerosis. In this study we examined the capacity of alphaB-crystallin or peptides derived thereof to induce experimental autoimmune encephalomyelitis (EAE) in SJL mice. Despite extensive efforts to induce EAE using active immunization with whole alphaB-crystallin, using adoptive transfer of lymphocytes or using peptide immunizations, no clinical or histological signs of EAE could be induced. SJL mice were unable to mount proliferative T-cell responses in vitro or delayed-type hypersensitivity responses in vivo to self-alphaB-crystallin. Also, immunization with self-alphaB-crystallin did not lead to any antibody response in SJL mice while bovine alphaB-crystallin triggered clear antibody responses within 1 week. Immunizations with alphaB-crystallin-derived peptides led to the activation of IL-4-producing Th2 cells and only a few IFN-gamma-producing Th1 cells. Peptide-specific T cells showed no cross-reactivity against whole alphaB-crystallin. The inability of SJL mice to mount proinflammatory T-cell responses against self-alphaB-crystallin readily explains the lack of EAE induction by immunization with whole protein or peptides derived from it. T- and B-cell nonresponsiveness is associated with constitutive expression of full-length alphaB-crystallin in both primary and secondary lymphoid organs in SJL mice, which is seen in other mammals as well, but not in humans.     

Radioautographic study of the cellular proliferation of retinal pigment epithelium in axolotls

The proliferative activity of the pigment epithelium cells in the axolotl eyes was studied using 3H-thymidine in two types experiments: after the removal of lens, iris and retina and upon the cultivation of the pigment epithelium pieces in the cavity of lens-less eye. Irrespective of the operation type, the level of proliferation of the pigment epithelium cells changed regularly with respect to the time of observation. In the intact eye, the level of proliferation of the pigment epithelium cells was not high: the index of labelled nuclei equaled 0.5%, no mitoses were found. The highest values of the index of labelled nuclei (12.6-32.1%) and of the mitotic index (0.54-1.07%) were registered on the 10-20th days after the operation. After 40 days, the indices of proliferative activity of the pigment epithelium cells approached gradually those for the intact eye. The cultivation of the pigment epithelium cells in the cavity of a lens-less eye for 50 days did not result in their transdifferentiation into retina cells. The layered retina found in 7.7% of cases after the removal of lens, iris and retina could regenerate either from the cells of the retina growth zone localized in the region of embryonic split, or due to transdifferentiation of the pigment epithelium cells.