Cellular and humoral immune responsiveness to inactivated Leptospira interrogans in dogs vaccinated with a tetravalent Leptospira vaccine.

Vaccination is commonly used to protect dogs against leptospirosis, however, memory immune responses induced by canine Leptospira vaccines have not been studied. In the present study, antibody and T cell mediated responses were assessed in dogs before and 2 weeks after annual revaccination with a commercial tetravalent Leptospira vaccine containing serogroups Canicola and Australis. Vaccination significantly increased average log2 IgG titers from 6.50 to 8.41 in year 1, from 5.99 to 7.32 in year 2, from 5.32 to 8.32 in year 3 and from 5.32 to 7.82 in year 4. The CXCL-10 levels, induced by in vitro stimulation of PBMC with Canicola and Australis, respectively, significantly increased from 1039.05 pg/ml and 1037.38 pg/ml before vaccination to 2547.73 pg/ml and 2730.38 pg/ml after vaccination. IFN-γ levels increased from 85.60 pg/ml and 178.13 pg/ml before vaccination to 538.62 pg/ml and 210.97 pg/ml after vaccination. The percentage of proliferating CD4+ T cells in response to respective Leptospira strains significantly increased from 1.43 % and 1.25 % before vaccination to 24.11 % and 14.64 % after vaccination. Similar responses were also found in the CD8+ T cell subset. Vaccination also significantly enhanced the percentages of central memory CD4+ T cells from 12 % to 26.97 % and 27.65 %, central memory CD8+ T cells from 3 % to 9.47 % and 7.55 %, and effector CD8+ T cells from 3 % to 7.6 % and 6.42 %, as defined by the expression of CD45RA and CD62L, following stimulation with Canicola and Australis, respectively. Lastly, enhanced expression of the activation marker CD25 on T cells after vaccination was found. Together, our results show that next to IgG responses, also T cell responses are induced in dogs upon annual revaccination with a tetravalent Leptospira vaccine, potentially contributing to protection.

Generation of the First TCR Transgenic Mouse with CD4(+) T Cells Recognizing an Anti-inflammatory Regulatory T Cell-Inducing Hsp70 Peptide.

Antigen-specific regulatory T cells (Tregs) directed at self-antigens are difficult to study since suitable specific tools to isolate and characterize these cells are lacking. A T cell receptor (TCR)-transgenic mouse would generate possibilities to study such -antigen-specific T cells. As was shown previously, immunization with the mycobacterial heat shock protein (Hsp) 70-derived peptide B29 and its mouse homologs mB29a and mB29b induced anti-inflammatory responses. Furthermore, B29 induced antigen--specific Tregs in vivo. To study mB29b-specific Tregs, we isolated the TCR from T cell hybridomas generated against mB29b and produced a TCR transgenic mouse that expresses a MHC-class II restricted mB29b-specific TCR. These TCR transgenic CD4(+) T cells were found to cross-react with the B29 epitope as identified with peptide-induced proliferation and IL-2 production. Thus, we have successfully generated a novel mouse model with antigen-specific CD4(+) T cells that recognize self and bacterial Hsp 70-derived peptides. With this novel mouse model, it will be possible to study primary antigen-specific T cells with specificity for a regulatory Hsp70 T cell epitope. This will enable the isolation and characterization CD4(+)CD25(+) Tregs with a proven specificity. This will provide useful knowledge of the induction, activation, and mode of action of Hsp70-specific Tregs, for instance, during experimental arthritis.

An Arthritis-Suppressive and Treg Cell-Inducing CD4+ T Cell Epitope Is Functional in the Context of HLA-Restricted T Cell Responses.

OBJECTIVE: We previously showed that mycobacterial Hsp70-derived peptide B29 induced B29-specific Treg cells that suppressed experimental arthritis in mice via cross-recognition of their mammalian Hsp70 homologs. The aim of the current study was to characterize B29 binding and specific CD4+ T cell responses in the context of human major histocompatibility complex (MHC) molecules. METHODS: Competitive binding assays were performed to examine binding of peptide B29 and its mammalian homologs to HLA molecules. The effect of B29 immunization in HLA-DQ8-transgenic mice with proteoglycan-induced arthritis was assessed, followed by ex vivo restimulation with B29 to examine the T cell response. Human peripheral blood mononuclear cells were used to investigate the presence of B29-specific T cells with immunoregulatory potential. RESULTS: The binding affinity of the B29 peptide was high to moderate for multiple HLA-DR and HLA-DQ molecules, including those highly associated with rheumatoid arthritis. This binding was considered to be functional, because B29 immunization resulted in the suppression of arthritis and T cell responses in HLA-DQ8-transgenic mice. In humans, we demonstrated the presence and expansion of B29-specific CD4+ T cells, which were cross-reactive with the mammalian homologs. Using HLA-DR4+ tetramers specific for B29 or the mammalian homolog mB29b, we showed expansion of cross-reactive T cells, especially the human FoxP3+ CD4+CD25+ T cell population, after in vitro stimulation with B29. CONCLUSION: These results demonstrated a conserved fine specificity and functionality of B29-induced Treg cell responses in the context of the human MHC. Based on these findings, a path for translation of the experimental findings for B29 into a clinical immunomodulatory therapeutic approach is within reach.

DEC205+ Dendritic Cell-Targeted Tolerogenic Vaccination Promotes Immune Tolerance in Experimental Autoimmune Arthritis.

Previous studies in mouse models of autoimmune diabetes and encephalomyelitis have indicated that the selective delivery of self-antigen to the endocytic receptor DEC205 on steady-state dendritic cells (DCs) may represent a suitable approach to induce Ag-specific immune tolerance. In this study, we aimed to examine whether DEC205(+) DC targeting of a single immunodominant peptide derived from human cartilage proteoglycan (PG) can promote immune tolerance in PG-induced arthritis (PGIA). Besides disease induction by immunization with whole PG protein with a high degree of antigenic complexity, PGIA substantially differs from previously studied autoimmune models not only in the target tissue of autoimmune destruction but also in the nature of pathogenic immune effector cells. Our results show that DEC205(+) DC targeting of the PG peptide 70-84 is sufficient to efficiently protect against PGIA development. Complementary mechanistic studies support a model in which DEC205(+) DC targeting leads to insufficient germinal center B cell support by PG-specific follicular helper T cells. Consequently, impaired germinal center formation results in lower Ab titers, severely compromising the development of PGIA. Overall, this study further corroborates the potential of prospective tolerogenic DEC205(+) DC vaccination to interfere with autoimmune diseases, such as rheumatoid arthritis.

Activated peritoneal cavity B-1a cells possess regulatory B cell properties.

Previous studies have suggested that murine peritoneal cavity-derived B-1a cells possess similarities with described regulatory B cell subsets. The aim of the current study was to examine the potential immunoregulatory function of peritoneal cavity-derived B(-1a) cells. In vitro activation of peritoneal cavity-derived B- and B-1a cells shows that activation of these B cells with anti-CD40 and LPS induces these cells to secrete more IL-10, IL-6 and IgM as compared to splenic B cells. In a suppression assay, CD40/TLR4-activated peritoneal cavity B cells possess regulatory B cell functions as they inhibit the capacity of CD4(+) T cells to produce both tumor necrosis factor-α and interferon-γ. Splenic B cells did not show this, whereas non-activated peritoneal cavity B cells augmented the capacity of CD4(+) T cells to produce tumor necrosis factor-α, while the ability to produce interferon-γ was not altered. The current paper compares splenic B cells to peritoneal cavity B(-1a) cells in an in vitro activation- and an suppression-assay and concludes that peritoneal cavity B(-1a) cells possess properties that appear similar to splenic autoimmune-suppressive regulatory B cell subsets described in the literature.

Effects of discontinuing anti-tumor necrosis factor therapy during pregnancy on the course of inflammatory bowel disease and neonatal exposure.

BACKGROUND & AIMS: We assessed the course of inflammatory bowel disease (IBD) among pregnant women who stopped taking anti-tumor necrosis factor (TNF) agents. We also analyzed levels of anti-TNF agents in cord blood samples. METHODS: We followed 31 pregnancies in 28 women with IBD between April 2006 and April 2011 who were treated with anti-TNF agents (18 received infliximab, and 13 received adalimumab) during pregnancy. We used enzyme-linked immunosorbent assays to measure levels of anti-TNF agents in cord blood collected from 18 newborns (12 whose mothers took infliximab, and 6 whose mothers took adalimumab). RESULTS: Among the patients taking infliximab, 12 (71%) discontinued treatment before gestational week 30; all patients remained in remission. All the patients taking adalimumab discontinued treatment before gestational week 30; two patients had relapses of IBD. There were 28 live births, 1 miscarriage among patients taking infliximab (at gestational week 6), and 2 miscarriages among patients taking adalimumab (at weeks 6 and 8); there were no congenital malformations. The mean cord blood level of infliximab was 6.4 ± 1.6 µg/mL; it was significantly lower among women who received the drug 10 weeks or less before delivery (2.8 ± 1.1 µg/mL) than those who received infliximab closer to delivery (10 ± 2.3 µg/mL; P = .02). Adalimumab was detected in 5 samples of cord blood (mean concentration, 1.7 ± 0.4 µg/mL); 1 cord blood sample from a woman who discontinued the treatment at gestational week 22 had an undetectable level of the drug. CONCLUSIONS: Discontinuation of anti-TNF therapy appears to be safe for pregnant women with quiescent IBD. However, these drugs are still detected in cord blood samples.

Regulatory T cells that recognize a ubiquitous stress-inducible self-antigen are long-lived suppressors of autoimmune arthritis.

Reestablishing self-tolerance in autoimmunity is thought to depend on self-reactive regulatory T cells (Tregs). Exploiting these antigen-specific regulators is hampered by the obscure nature of disease-relevant autoantigens. We have uncovered potent disease-suppressive Tregs recognizing Heat Shock Protein (Hsp) 70 self-antigens, enabling selective activity in inflamed tissues. Hsp70 is a major contributor to the MHC class II ligandome. Here we show that a conserved Hsp70 epitope (B29) is present in murine MHC class II and that upon transfer, B29-induced CD4(+)CD25(+)Foxp3(+) T cells suppress established proteoglycan-induced arthritis in mice. These self-antigen-specific Tregs were activated in vivo, and when using Lymphocyte Activation Gene-3 as a selection marker, as few as 4,000 cells sufficed. Furthermore, depletion of transferred Tregs abrogated disease suppression. Transferred cells exhibited a stable phenotype and were found in joints and draining lymph nodes up to 2 mo after transfer. Given that (i) B29 administration by itself suppressed disease, (ii) our findings were made with wild-type (T-cell receptor nontransgenic) Tregs, and (iii) the B29 human homolog is presented by HLA class II, we are nearing translation of antigen-specific Treg activation as a promising intervention for chronic inflammatory diseases.

Hsp70 vaccination-induced antibodies recognize B cell epitopes in the cell wall of Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic intestinal infection of ruminants and has been associated with the etiology of human Crohn's disease. A MAP Hsp70/DDA subunit vaccine previously showed a significant reduction in fecal shedding of MAP in cattle, concomitant with pronounced antibody production against MAP Hsp70, rather than T cell reactivity. Our hypothesis is that if Hsp70-specific antibodies are able to confer protection, the first requisite would be that the Hsp70 molecule is accessible for antibodies in intact MAP bacteria. In the current study monoclonal antibodies identified MAP Hsp70 B cell epitopes. Two linear epitopes were also recognized by antibodies of vaccinated calves and goats. These epitopes showed to be accessible by antibodies in the bacterial cell wall and in intestinal lesional tissue during natural infection. These results indicate that vaccination-induced antibodies can bind intact bacteria and have the potential to contribute to the protective effect of Hsp70/DDA subunit vaccination against bovine paratuberculosis.

Epitopes of Mycobacterium avium ssp. paratuberculosis 70kDa heat-shock protein activate bovine helper T cells in outbred cattle.

The recombinant 70kDa heat-shock protein of Mycobacterium avium subspecies paratuberculosis (MAP Hsp70) has been shown to be an immunodominant antigen and a subunit vaccine candidate for bovine paratuberculosis. The aim of the present study was to define MAP Hsp70 specific T cell epitopes in cows immunized with MAP Hsp70 and cows experimentally infected with MAP. Nine peptides were shown to induce proliferation and interferon-gamma production by lymphocytes from MAP Hsp70 immunized cattle. From 28 calves experimentally infected with MAP 82% responded to at least one of the 5 most immunodominant peptides, indicating relevance of the epitopes during infection. In these 28 animals 15 different BoLA class II haplotypes were present indicating that the peptides were presented by multiple BoLA class II DRB3 alleles. These findings indicate the potential of the MAP Hsp70 subunit vaccine as a tool to control paratuberculosis in outbred cattle populations.

Subpopulations of bovine WC1(+) gammadelta T cells rather than CD4(+)CD25(high) Foxp3(+) T cells act as immune regulatory cells ex vivo.

Regulatory T cells (Treg) are regarded essential components for maintenance of immune homeostasis. Especially CD4(+)CD25(high) T cells are considered to be important regulators of immune reactivity. In humans and rodents these natural Treg are characterized by their anergic nature, defined as a non-proliferative state, suppressive function and expression of Foxp3. In this study the potential functional role of flowcytometry-sorted bovine white blood cell populations, including CD4(+)CD25(high) T cells and gammadelta T cell subpopulations, as distinct ex vivo regulatory cells was assessed in co-culture suppression assays. Our findings revealed that despite the existence of a distinct bovine CD4(+)CD25(high) T cell population, which showed Foxp3 transcription/expression, natural regulatory activity did not reside in this cell population. In bovine co-culture suppression assays these cells were neither anergic nor suppressive. Subsequently, the following cell populations were tested functionally for regulatory activity: CD4(+)CD25(low) T cells, WC1(+), WC1.1(+) and WC1.2(+) gammadelta T cells, NK cells, CD8(+) T cells and CD14(+) monocytes. Only the WC1.1(+) and WC1.2(+) gammadelta T cells and CD14(+) monocytes proved to act as regulatory cells in cattle, which was supported by the fact that these regulatory cells showed IL-10 transcription/expression. In conclusion, our data provide first evidence that cattle CD4(+)CD25(high)Foxp3(+) and CD4(+)CD25(low) T cells do not function as Treg ex vivo. The bovine Treg function appears to reside in the gammadelta T cell population, more precisely in the WC1.1(+) and the WC1.2(+) subpopulation, major populations present in blood of cattle in contrast to non-ruminant species.

Energy metabolism of bloodstream form Trypanosoma theileri.

Bloodstream form Trypanosoma theileri degrades glucose to acetate (47%) and succinate (45%) and, therefore, does not solely rely on glycolysis for ATP production. This trypanosomatid does not use amino acids for energy metabolism. These results refute the prevailing hypothesis that substrate availability determines the type of energy metabolism of trypanosomatids.

Mycobacterial 70 kD heat-shock protein is an effective subunit vaccine against bovine paratuberculosis.

Paratuberculosis is a chronic granulomatous inflammation of the small intestine of cattle and other ruminants, caused by infection with Mycobacterium avium ssp. paratuberculosis (MAP). The disease can be found in ruminant herds worldwide, causing substantial economic losses at farm level due to premature culling and production losses. In previous studies, it has been shown that immune responses to recombinant MAP Hsp70 proteins were predominantly cell mediated. As protective immunity to the intracellular mycobacterial pathogens is thought to be cell-mediated in origin, we have studied the use of a recombinant MAP Hsp70 as a subunit vaccine in cattle experimentally infected with MAP. The results of the current study demonstrate that recombinant MAP Hsp70 can be successfully used as a subunit vaccine against bovine paratuberculosis, significantly reducing shedding of bacteria in feces during the first 2 years following experimental infection.

Comparison of three assays for the evaluation of specific cellular immunity to Leishmania infantum in dogs.

Cellular immune response to Leishmania plays a key role in canine leishmaniosis. However, there are few assays to evaluate this response in the dog. Here, we evaluated and compared three assays of specific cellular immune response to Leishmania infantum in dogs: the leishmanin skin test (LST), lymphocyte proliferation assay (LPA) and IFN-gamma cytopathic effect inhibition bioassay (IFNB). Fifty-six healthy dogs from an endemic area for leishmaniosis on the island of Mallorca were studied. In all, 37 dogs showed a positive LST and 32 a positive IFNB, 24 were positive for both assays. The 17 dogs positive for LPA also gave positive results for either LST or IFNB, or both. These findings indicate that although LST is the method of choice, IFNB is a complementary test. Therefore, both assays should be performed and analyzed jointly. In comparison with LST and IFNB, LPA is much less sensitive, and yields many false negative results.

The effects of a single injection of dexamethasone-21-isonicotinate on the lymphocyte functions of dairy cows at two weeks post partum.

Dexamethasone is a potent therapeutic for treatment of the fatty liver syndrome or primary ketosis in post partum dairy cows. Reservations exist, however, among practitioners with respect to the risk of immunosuppression induced by corticosteroids. The aim of this study was to investigate the effect of a single injection of dexamethasone-21-isonicotinate on distinct immune functions of postpartum dairy cows because only scarce information is available on the effects of corticosteroid preparations when administered at a dosage and frequency for treatment of the fatty liver syndrome or primary ketosis. Sixteen Swedish red-pied dairy cows, between days 9 and 15 post partum, were allotted to either a control group (n = 8) or a treatment group (n = 8). The cows in the treatment group received a single intramuscular injection of a dexamethasone-21-isonicotinate suspension at a dosage of 0.02 mg/kg i.m. at the start of the experiment. White blood cell counts and selected lymphocyte functions (lymphocyte proliferation, expression of lymphocyte markers and the b2 and a4 chain of adhesion molecules belonging to the integrin family) and some parameters of the energy metabolism (glucose, insulin) were determined before the administration of corticosteroids (day 0) and subsequently at days 2, 4, 7 and 9 of the experiment. Changes in glucose and insulin were within the target range for treatment of the fatty liver syndrome or primary ketosis. Significant (P < 0.05) increases in the number of circulating white blood cells were observed in treated cows on the second day following treatment which was exclusively caused by an increase in the number of circulating neutrophils. Lymphocyte blastogenesis in response to ConA and the percentages of lymphocytes positive for CD2, CD4, CD8, CD49d and CD18 as well as the intensity of CD49d expression did not differ between the treatment and control groups. There was, however, a significant reduction (P < 0.01) in the intensity of CD18 expression on lymphocytes in the treated animals on the fourth day after treatment. In conclusion, a single administration of dexamethasone-21-isonicotinate in a dosage of 0.02 mg/kg i.m. at two weeks post partum in healthy cows had a significant but highly transient effect on CD18 expression on lymphocytes and the number of peripheral blood neutrophils, but did not affect lymphocyte blastogenesis or lymphocyte subpopulation patterns in peripheral blood.

Progressive bovine paratuberculosis is associated with local loss of CD4(+) T cells, increased frequency of gamma delta T cells, and related changes in T-cell function.

Bovine paratuberculosis is caused by the infection of young calves with Mycobacterium avium subsp. paratuberculosis, resulting in a chronic granulomatous infection of predominantly the ileum. After an incubation period of 2 to 5 years, the disease becomes progressive in some of the chronically infected, but asymptomatic cows. This results in a protein-losing enteropathy that will ultimately be fatal. A loss of cell-mediated immune responses in symptomatic animals has been described, but no information is available concerning immune reactivity in the intestine. We sought to investigate putative disease status-associated lymphocyte subset distributions and antigen-specific functional characteristics of mononuclear cells isolated from blood, gut-associated lymphoid tissue, and the intestinal walls of 22 cows in different stages of disease and in control animals. The results demonstrated a significant decrease in CD4(+) T-cell frequency and a significant increase in TcR1-N12(+) gamma delta T-cell frequency in ileum lamina propria lymphocytes of symptomatic animals compared to the asymptomatic shedders. Immunohistology revealed that there was also an absolute decrease in the number of CD4(+) T cells in sections of the lesional ileum. Our findings also indicated that both peripheral and intestinal cell-mediated responses are decreased in symptomatic animals compared to asymptomatic animals. We conclude that the decrease in cell-mediated responses is likely related to a loss of antigen-specific CD4(+) T cells, which is most prominent in the lesional ileum from symptomatic animals, thus contributing to the progressive nature of bovine paratuberculosis.