Oleg S. Targoni

Protective Anti‐Helicobacter Immunity Is Induced with Aluminum Hydroxide or Complete Freund’s Adjuvant by Systemic Immunization

To determine whether systemic immunization against Helicobacter pylori could be achieved with an adjuvant approved for human use, the efficacy of vaccination with Helicobacter antigen in combination with aluminum hydroxide (AlOH) was evaluated in a murine model of Helicobacter infection. Immunization with antigen and AlOH induced interleukin-5-secreting, antigen-specific T cells, and immunization with antigen and complete Freunds adjuvant induced interferon-gamma-secreting, antigen-specific T cells, as determined by ELISPOT assay. Both immune responses conferred protection after challenge with either H. pylori or H. felis, as confirmed by the complete absence of any bacteria, as assessed by both histology and culture of gastric biopsy samples. Protection was antibody independent, as demonstrated with antibody-deficient muMT mice (immunoglobulin-gene knockout mice), and CD4(+) spleen T cells from immunized mice were sufficient to transfer protective immunity to otherwise immunodeficient rag1(-/-) recipients. These results suggest an alternative and potentially more expeditious strategy for development of a human-use H. pylori vaccine.

Frequencies of neuroantigen-specific T cells in the central nervous system versus the immune periphery during the course of experimental allergic encephalomyelitis.

Direct measurements of the frequency and the cytokine signature of the neuroantigen-specific effector cells in experimental allergic encephalomyelitis (EAE) are a continuing challenge. This is true for lymphoid tissues, and more importantly, for the CNS itself. Using enzyme-linked immunospot analysis (ELISPOT) assays, we followed proteolipid protein (PLP) 139–151-specific T cells engaged by active immunization of SJL mice. The total numbers of PLP139–151-specific CD4 cells were highest before disease onset. At this time, these cells resided in lymphoid and nonlymphoid tissues, but were not detected in the CNS. While the PLP139–151-specific cells reached high frequencies in the CNS during clinical EAE, in absolute numbers, less than 20% of them were present in the target organ, with the majority residing in the periphery throughout all stages of the disease. The numbers of PLP139–151-specific cells gradually declined in both compartments with time. While eventually this first wave of effector cells completely disappeared from the CNS, PLP178–191-specific cells became engaged, being detected first in the CNS. These data suggest that throughout all stages of EAE, the effector cells in the CNS are recruited from a vast peripheral reservoir, and that the second wave of effector cells is engaged while the first wave undergoes exhaustion.

Detection of low-frequency antigen-specific IL-10-producing CD4 + T cells via ELISPOT in PBMC: cognate vs. nonspecific production of the cytokine

Single-cell resolution cytokine ELISPOT assays are increasingly used to gain insights into clonal sizes of type 1 and type 2 effector T cell populations in vivo. However, ELISPOT assays permitting monitoring of regulatory IL-10-producing T cells have so far not been established. Unlike IFN-gamma, IL-2, IL-4, and IL-5 assays performed on PBMC in which the recall antigen-induced cytokine spots are T cell-derived, we show here that in such assays IL-10 is primarily monocyte-derived. T cell-derived IL-10 spots were 80 x 10(3) microm(2) in size, seven times larger than spots produced by monocytes, and B cells produced even smaller spots. Based on spot size gating and the use of B cells as APC, we have established test conditions that permit measurement of cognate IL-10 production by low-frequency antigen-specific T cells. IL-10-producing PPD-specific CD4(+) T cells were detected in frequencies comparable to IFN-gamma-secreting CD4(+) T cells in tuberculosis patients, but not in uninfected healthy control individuals. In contrast, IL-10-secreting CD4(+) T cells specific for a panel of recall antigens could not be detected in frequencies >1/100,000 in healthy individuals whose CD4(+) cells responded to these antigens with type 1 or type 2 cytokine production in the 1:100,000-1:1000 frequency range. Therefore, the induction of IL-10-producing T cells seems to be under tighter control than that of Th1/Th2 cells, apparently confined to states of chronic immune stimulation. Access to low-frequency immune monitoring of IL-10-producing T cells will provide new insights into the role of regulatory T cells in health and disease.

Shifting T-cell activation thresholds in autoimmunity and determinant spreading

Summary: The best‐characterized autoimmune T‐cell response is that to myelin basic protein (MBP), MBP has classically been regarded as a sequestered antigen that does not cause negative selection. This view has been fostered by the observation that T‐cell receptor‐transgenic T cells that are specific for the “immunodominant determinant” on the molecule, MBP:Ac 1–11, persist as naive cells in MBP‐expressing H‐Z” mice. The same T cells, however, can cause autoimmune pathology once they have been primed by environmental stimulation to become memory cells. Once the autoimmune response to Ac1‐11 has been engaged, determinant spreading occurs and second‐wave T‐cell responses that are specific for weaker, “cryptic” determinants like MBP: 121–140 develop. Although the nature of these cryptic determinants has been enigmatic, recent studies using MBP‐”‐mice have provided new insights. These studies showed that MBP is not a sequestered antigen, but one that causes negative selection; as MBP: 121–140 is actually the immunodominant determinant in MBP‐/‐ mice, it tolerizes high avidity clones in MBP+/+ mice, making it appear cryptic. Based on this new information, we attempt here to redefine the MBP‐specific repertoire within the theoretical framework of the threshold model for negative selection, and we propose a model of shifting T‐cell activation thresholds to explain how ignorant/naive T cells can become effector cells of autoimmune pathology and why this effector cell repertoire spreads.

Does the Frequency and Avidity Spectrum of the Neuroantigen-Specific T Cells in the Blood Mirror the Autoimmune Process in the Central Nervous System of Mice Undergoing Experimental Allergic Encephalomyelitis?

In humans, studies of autoreactive T cells that mediate multiple sclerosis have been largely confined to testing peripheral blood lymphocytes. Little is known how such measurements reflect the disease-mediating autoreactive T cells in the CNS. This information is also not available for murine experimental allergic encephalomyelitis (EAE); the low number of T cells that can be obtained from the blood or the brain of mice prevented such comparisons. We used single-cell resolution IFN-γ ELISPOT assays to measure the frequencies and functional avidities of myelin basic protein (MBP:87–99)-specific CD4 cells in SJL mice immunized with this peptide. Functional MBP:87–99-specific IFN-γ-producing cells were present in the CNS during clinical signs of EAE, but not during phases of recovery. In contrast, MBP:87–99-specific T cells persisted in the blood during all stages of the disease, and were also present in mice that did not develop EAE. Therefore, the increased frequency of MBP:87–99-reactive T cells in the blood reliably reflected the primed state, but not the inflammatory activity of these cells in the brain. The functional avidity of the MBP:87–99-reactive T cells was identical in the brain and blood and did not change over 2 mo as the mice progressed from acute to chronic EAE. Therefore, high-affinity T cells did not become selectively enriched in the target organ, and avidity maturation of the MBP:87–99-specific T cell repertoire did not occur in the observation period. The data may help the interpretation of measurements made with peripheral blood lymphocytes of multiple sclerosis patients.

Increased per cell IFN-γ productivity indicates recent in vivo activation of T cells

Immunization with vaccinia virus causes long-term immunity. Efforts have been made to characterize the T cells responsible for this protection. Recently, T cell subsets were described that not only co-express multiple cytokines, but also show increased per cell cytokine productivity. These highly productive cells are often considered to be the most protective. We used ELISPOT assays to measure per cell IFN-gamma productivity of vaccinia-specific T cells in childhood immunized adults immediately before and at different time points after vaccinia re-vaccination. Apart from an increase in frequency, we found a marked increase of IFN-gamma productivity following vaccinia re-vaccination. However, these changes were short-lived as both parameters quickly returned to baseline values within 22days after re-vaccination. Therefore, increased per cell IFN-gamma productivity seems to be a sign of recent in vivo T cell activation rather than a stable marker of a distinct T cell subset responsible for long-term immune protection.

Immunogenicity of unprocessed and photooxidized bovine and human osteochondral grafts in collagen-sensitive mice

BackgroundAutologous and allogeneic osteochondral grafts have been used to repair damaged or diseased cartilage. There are drawbacks to both of these methods, however. Another possible source for osteochondral grafting is photooxidized xenograft scaffolds. The purpose of this study was to evaluate the adaptive immune response to unprocessed and photooxidized xenogeneic osteochondral grafts in a collagen-sensitive mouse model.MethodsUnprocessed and photooxidized bovine and human osteochondral grafts were used. The grafts were implanted subcutaneously in collagen-sensitive DBA/1LacJ mice for four or twelve weeks. ELISPOT assays were conducted with spleen cells to evaluate the number of collagen-specific T cells that produce IL-2, IL-4, IL-5 or IFN-γ. Serum was collected and ELISA assays were performed to determine the titers of collagen-specific and total IgG, IgG1, IgG2a, or IgM antibodies. Histology was conducted on the retrieved osteochondral grafts.ResultsResults indicated that, with respect to adaptive T cell immunity, the photooxidized bovine grafts, unprocessed human grafts and photooxidized human grafts did not induce a significant response to collagen. The unprocessed bovine grafts, however, were slightly more immunogenic, inducing a weak immune response. With respect to antibody production, the bovine grafts were less immunogenic than the human grafts. Bovine collagen-specific IgG antibodies were not induced by these grafts, but production of IgM after twelve weeks was observed with both the unprocessed and photooxidized bovine grafts. In contrast, photooxidized human osteochondral grafts induced IgG1 and IgG2a antibodies, while the unprocessed human grafts did not. Pre-existing human collagen-specific IgM antibodies were present in all mice, including sham-operated negative controls that did not receive an implant. Histological analysis revealed some degree of fibrous encapsulation and inflammatory infiltrations in both bovine and human implants, whether unprocessed or photooxidized.ConclusionBoth bovine and human cartilage grafts showed weak, but clear immunogenicity in the DBA/1LacJ mice, indicating that immunogenic collagen was still contained in the grafts, even after cleaning and photooxidation. The process of photooxidation is still important in osteochondral grafting, since it stabilizes the surface of the cartilage by cross-linking the collagen fibers, and allows for immediate load bearing and joint resurfacing.

Immunologic testing of xeno-derived osteochondral grafts using peripheral blood mononuclear cells from healthy human donors

BackgroundOne means of treating osteoarthritis is with autologous or allogeneic osteochondral grafts. The purpose of this study was to evaluate the innate immunological response in humans toward xeno-derived osteochondral grafts that have been partially or entirely treated by the photooxidation process.MethodsThe antigens tested included bovine, porcine, ovine and equine osteochondral samples that have been treated in successive steps of photooxidation. ELISPOT assays were used to evaluate the production of IL-1, IL-4, IL-6, IL-10, IL-12 and TNF-α by human monocytes in response to the antigens.ResultsResults indicated vigorous production of IL-1, IL-6, IL-10 and TNF-α in response to untreated bovine, porcine and equine specimens. This indicates that these samples are perceived as foreign, or stimulatory, by the human monocytes. There was no induction of IL-4 or IL-12, which is required for Th2 and Th1 immunity, respectively. In contrast, the processed bovine, porcine and equine samples did not induce significant activation of cells of the innate immune system. This occurred after the first step in processing (after cleaning in increasing strengths of ethanol). This suggests that the processing steps dramatically, if not completely, negated the immunostimulatory properties of the test sample. The results for the ovine samples indicate a reverse response.ConclusionThe findings of the study suggest that photooxidized bovine, porcine or equine samples have the potential to be used as an osteochondral graft. Although the first step in processing reduced the immunological response, photooxidation is still necessary to retain the structure and mechanical integrity of the cartilage, which would allow for immediate joint resurfacing.

How much of Virus-Specific CD8 T Cell Reactivity is Detected with a Peptide Pool when Compared to Individual Peptides?

Immune monitoring of T cell responses increasingly relies on the use of peptide pools. Peptides, when restricted by the same HLA allele, and presented from within the same peptide pool, can compete for HLA binding sites. What impact such competition has on functional T cell stimulation, however, is not clear. Using a model peptide pool that is comprised of 32 well-defined viral epitopes from Cytomegalovirus, Epstein-Barr virus, and Influenza viruses (CEF peptide pool), we assessed peptide competition in PBMC from 42 human subjects. The magnitude of the peptide pool-elicited CD8 T cell responses was a mean 79% and a median 77% of the sum of the CD8 T cell responses elicited by the individual peptides. Therefore, while the effect of peptide competition was evident, it was of a relatively minor magnitude. By studying the dose-response curves for individual CEF peptides, we show that several of these peptides are present in the CEF-pool at concentrations that are orders of magnitude in excess of what is needed for the activation threshold of the CD8 T cells. The presence of such T cells with very high functional avidity for the viral antigens can explain why the effect of peptide competition is relatively minor within the CEF-pool.

Inhibition of mitogen-stimulated proliferation of human lymphocytesin vitro by synthetic peptide fragments of α-2 interferon

Human α-2 interferon and peptides representing parts of the amino-acid sequence 124–138 of the IF molecule inhibit the proliferative response of peripheral blood lymphocytes from healthy donorsin vitro induced by ConA. It is shown that neither interferon α-2 nor biologically active peptides change the level of interleukin-2 ConA-induced production by human blood mononuclears.