Mice from a genetically resistant background lacking the interferon gamma receptor are susceptible to infection with Leishmania major but mount a polarized T helper cell 1-type CD4+ T cell response.

Mice with homologous disruption of the gene coding for the ligand-binding chain of the interferon (IFN) gamma receptor and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in the differentiation of functional CD4+ T cell subsets in vivo and resistance to infection. Wild-type 129/Sv/Ev mice are resistant to infection with this parasite, developing only small lesions, which resolve spontaneously within 6 wk. In contrast, mice lacking the IFN-gamma receptor develop large, progressing lesions. After infection, lymph nodes (LN) and spleens from both wild-type and knockout mice showed an expansion of CD4+ cells producing IFN-gamma as revealed by measuring IFN-gamma in supernatants of specifically stimulated CD4+ T cells, by enumerating IFN-gamma-producing T cells, and by Northern blot analysis of IFN-gamma transcripts. No biologically active interleukin (IL) 4 was detected in supernatants of in vitro-stimulated LN or spleen cells from infected wild-type or deficient mice. Reverse transcription polymerase chain reaction analysis with primers specific for IL-4 showed similar IL-4 message levels in LN from both types of mice. The IL-4 message levels observed were comparable to those found in similarly infected C57BL/6 mice and significantly lower than the levels found in BALB/c mice. Anti-IFN-gamma treatment of both types of mice failed to alter the pattern of cytokines produced after infection. These data show that even in the absence of IFN-gamma receptors, T helper cell (Th) 1-type responses still develop in genetically resistant mice with no evidence for the expansion of Th2 cells.

RDM1, a novel RNA recognition motif (RRM)-containing protein involved in the cell response to cisplatin in vertebrates.

A variety of cellular proteins has the ability to recognize DNA lesions induced by the anti-cancer drug cisplatin, with diverse consequences on their repair and on the therapeutic effectiveness of this drug. We report a novel gene involved in the cell response to cisplatin in vertebrates. The RDM1 gene (for RAD52 Motif 1) was identified while searching databases for sequences showing similarities to RAD52, a protein involved in homologous recombination and DNA double-strand break repair. Ablation of RDM1 in the chicken B cell line DT40 led to a more than 3-fold increase in sensitivity to cisplatin. However, RDM1-/- cells were not hypersensitive to DNA damages caused by ionizing radiation, UV irradiation, or the alkylating agent methylmethane sulfonate. The RDM1 protein displays a nucleic acid binding domain of the RNA recognition motif (RRM) type. By using gel-shift assays and electron microscopy, we show that purified, recombinant chicken RDM1 protein interacts with single-stranded DNA as well as double-stranded DNA, on which it assembles filament-like structures. Notably, RDM1 recognizes DNA distortions induced by cisplatin-DNA adducts in vitro. Finally, human RDM1 transcripts are abundant in the testis, suggesting a possible role during spermatogenesis.

The VZV/IE63-specific T cell response prevents herpes zoster in fingolimod-treated patients.

OBJECTIVE: To assess longitudinally the antiviral immune response of T cells from patients with multiple sclerosis (MS) treated with fingolimod (FTY) vs other disease-modifying treatments (DMTs). METHODS: We assessed cellular immune responses specific to influenza virus (FLU), JC virus (JCV), and varicella-zoster virus (VZV) using quantification of interferon-γ secretion by enzyme-linked immunospot in patients with MS on FTY (n = 31), including 2 with herpes zoster (HZ), natalizumab (n = 11), and other DMTs (n = 11). We used viral lysates for FLU and VZV and a pool of peptides for FLU, JCV (VP-1), and VZV (IE63). RESULTS: Besides an expected drop of T cells, we found that, proportionally to the number of CD3(+) T cells, only FTY-treated patients with MS exhibited an increased VZV/IE63-specific T cell response peaking 6 months into treatment, a response that returned to baseline after 12 and 24 months. Two FTY-treated patients developed an HZ 6 months into treatment, coinciding with an absent VZV/IE63-specific T cell response. However, cellular immune responses specific to VZV lysate, JCV, and FLU (lysate and pool of peptide epitopes) were similar between all 3 categories (FTY, natalizumab, and other DMTs) of study patients. CONCLUSIONS: FTY-treated patients with MS exhibit an increased VZV/IE63-specific cellular immune response after 6 months of treatment. FTY-treated patients who develop an HZ are not able to mount such a response, suggesting that a T cell response directed against this viral protein may be key in preventing the occurrence of HZ.

Regulation of T cell response to leishmania antigens by determinants of histocompatibility leukocyte class I and II molecules

It has been shown that HLA class I molecules play a significant role in the regulation of the proliferation of T cells activated by mitogens and antigens. We evaluated the ability of mAb to a framework determinant of HLA class I molecules to regulate T cell proliferation and interferon gamma (IFN-g) production against leishmania, PPD, C. albicans and tetanus toxoid antigens in patients with tegumentary leishmaniasis and healthy subjects. The anti-major histocompatibility complex (MHC) mAb (W6/32) suppressed lymphocyte proliferation by 90% in cultures stimulated with aCD3, but the suppression was variable in cultures stimulated with leishmania antigen. This suppression ranged from 30-67% and was observed only in 5 of 11 patients. IFN-g production against leishmania antigen was also suppressed by anti-HLA class I mAb. In 3 patients IFN-g levels were suppressed by more than 60%, while in the other 2 cultures IFN-g levels were 36 and 10% lower than controls. The suppression by HLA class I mAb to the proliferative response in leishmaniasis patients and in healthy controls varied with the antigens and the patients or donors tested. To determine whether the suppression is directed at antigen presenting cells (APCs) or at the responding T cells, experiments with antigen-primed non-adherent cells, separately incubated with W6/32, were performed. Suppression of proliferation was only observed when the W6/32 mAb was added in the presence of T cells. These data provide evidence that a mAb directed at HLA class I framework determinants can suppress proliferation and cytokine secretion in response to several antigens.

Ageing impairs the T cell response to dendritic cells

Dendritic cells (DCs) are critical in priming adaptive T-cell responses, but the effects of ageing on interactions between DCs and T cells are unclear. This study investigated the influence of ageing on the maturation of and cytokine production by human blood-enriched DCs, and the impact on T cell responses in an allogeneic mixed leucocyte reaction (MLR). DCs from old subjects (65-75y) produced significantly less TNF-α and IFN-γ than young subjects (20-30y) in response to lipopolysaccharide (LPS), but expression of maturation markers and co-stimulatory molecules was preserved. In the MLR, DCs from older subjects induced significantly restricted proliferation of young T cells, activation of CD8+ T cells and expression of IL-12 and IFN-γ in T cells compared with young DCs. T cells from older subjects responded more weakly to DC stimulation compared with young T cells, regardless of whether the DCs were derived from young or older subjects. In conclusion, the capacity of DCs to induce T cell activation is significantly impaired by ageing.

Kinetics of B cell response during Yersinia enterocolitica infection in resistant and susceptible strains of mice

In this study we analyze the B-cell response in murine yersiniosis. To this end, we determined whether polyclonal activation of B-lymphocytes occurs during infection of susceptible (BALB/c) and resistant (C57BL/6) mice with Y. enterocolitica 0:8 and compared the immunoglobulin (Ig) isotypes produced in response to the infection by the two strains. The number of splenic cells secreting nonspecific and specific immunoglobulins was determined by ELISPOT. The presence of anti-Yersinia antibodies in serum was detected by ELISA. In both strains, the number of specific Ig-secreting cells was relatively low. Polyclonal B-cell activation was observed in both strains of mice, and the greatest activation was observed in the BALB/c mice, mainly for lgG(1)- and IgG(3)- secreting cells. The C57BL/6 mice showed a predominance of IgG(2a)-secreting cells. The peak production of anti-Yersinia IgG antibodies in the sera of BALB/c mice was seen on the 28th day after infection. The greatest increase in IgM occurred on the 14th day. A progressive increase of specific IgG antibodies was observed in C57BL/6 mice up to the 28th day after infection while IgM increased on the 21st day after infection. The production of specific IgA antibodies was not detected in either BALB/c or C57BL/6 mice. We conclude that polyclonal. activation of B lymphocytes occurs in both the Yersinia resistant and Yersinia-susceptible mice and that the more intense activation of B lymphocytes observed in the susceptible BALB/c mice does not enhance their resistance to Y. enterocolitica infection.

Low-Level Laser Therapy Influences Mouse Odontoblast-Like Cell Response in Vitro

Objective: The purpose of this study was to analyze the influence of two different irradiation times with 85mW/cm(2) 830nm laser on the behavior of mouse odontoblast-like cells. Background data: The use of low-level laser therapy (LLLT) to stimulate pulp tissue is a reality, but few reports relate odontoblastic responses to irradiation in in vitro models. Methods: Odontoblast-like cells (MDPC-23) were cultivated and divided into three groups: control/nonirradiated (group 1); or irradiated with 85mW/cm(2), 830nm laser for 10 sec (0.8 J/cm(2)) (group 2); or for 50 sec (4.2 J/cm(2)) (group 3) with a wavelength of 830 nm. After 3, 7, and 10 days, it was analyzed: growth curve and cell viability, total protein content, alkaline phosphatase (ALP) activity, calcified nodules detection and quantification, collagen immunolocalization, vascular endothelial growth factor (VEGF) expression, and real-time polymerase chain reaction (PCR) for DMP1 gene. Data were analyzed by Kruskall-Wallis test (alpha = 0.05). Results: Cell growth was smaller in group 2 (p < 0.01), whereas viability was similar in all groups and at all periods. Total protein content and ALP activity increased on the 10th day with 0.8 J/cm(2) (p < 0.01), as well as the detection and quantification of mineralization nodules (p < 0.05), collagen, and VEGF expression (p < 0.01). The expression of DMP1 increased in all groups (p < 0.05) compared with control at 3 days, except for 0.8 J/cm(2) at 3 days and control at 10 days. Conclusions: LLLT influenced the behavior of odontoblast-like cells; the shorter time/smallest energy density promoted the expression of odontoblastic phenotype in a more significant way.

Anti-IL-2 Treatment Impairs the Expansion of T-reg Cell Population during Acute Malaria and Enhances the Th1 Cell Response at the Chronic Disease

Plasmodium chabaudi infection induces a rapid and intense splenic CD4(+) T cell response that contributes to both disease pathogenesis and the control of acute parasitemia. The subsequent development of clinical immunity to disease occurs concomitantly with the persistence of low levels of chronic parasitemia. The suppressive activity of regulatory T (T-reg) cells has been implicated in both development of clinical immunity and parasite persistence. To evaluate whether IL-2 is required to induce and to sustain the suppressive activity of T-reg cells in malaria, we examined in detail the effects of anti-IL-2 treatment with JES6-1 monoclonal antibody (mAb) on the splenic CD4(+) T cell response during acute and chronic P. chabaudi AS infection in C57BL/6 mice. JES6-1 treatment on days 0, 2 and 4 of infection partially inhibits the expansion of the CD4(+)CD25(+)Foxp3(+) cell population during acute malaria. Despite the concomitant secretion of IL-2 and expression of high affinity IL-2 receptor by large CD4(+) T cells, JES6-1 treatment does not impair effector CD4+ T cell activation and IFN-gamma production. However, at the chronic phase of the disease, an enhancement of cellular and humoral responses occurs in JES6-1-treated mice, with increased production of TNF-alpha and parasite-specific IgG2a antibodies. Furthermore, JES6-1 mAb completely blocked the in vitro proliferation of CD4(+) T cells from non-treated chronic mice, while it further increased the response of CD4(+) T cells from JES6-1-treated chronic mice. We conclude that JES6-1 treatment impairs the expansion of T-reg cell population during early P. chabaudi malaria and enhances the Th1 cell response in the late phase of the disease.

Pathogen-Induced Proapoptotic Phenotype and High CD95 (Fas) Expression Accompany a Suboptimal CD8(+) T-Cell Response: Reversal by Adenoviral Vaccine

MHC class la-restricted CD8(+) T cells are important mediators of the adaptive immune response against infections caused by intracellular microorganisms. Whereas antigen-specific effector CD8(+) T cells can clear infection caused by intracellular pathogens, in some circumstances, the immune response is suboptimal and the microorganisms survive, causing host death or chronic infection. Here, we explored the cellular and molecular mechanisms that could explain why CD8(+) T-cell-mediated immunity during infection with the human protozoan parasite Trypanosoma cruzi is not optimal. For that purpose, we compared the CD8(+) T-cell mediated immune responses in mice infected with T. cruzi or vaccinated with a recombinant adenovirus expressing an immunodominant parasite antigen. Several functional and phenotypic characteristics of specific CD8(+) T cells overlapped. Among few exceptions was an accelerated expansion of the immune response in adenoviral vaccinated mice when compared to infected ones. Also, there was an upregulated expression of the apoptotic-signaling receptor CD95 on the surface of specific T cells from infected mice, which was not observed in the case of adenoviral-vaccinated mice. Most importantly, adenoviral vaccine provided at the time of infection significantly reduced the upregulation of CD95 expression and the proapoptotic phenotype of pathogen-specific CD8(+) cells expanded during infection. In parallel, infected adenovirus-vaccinated mice had a stronger CD8(+) T-cell mediated immune response and survived an otherwise lethal infection. We concluded that a suboptimal CD8(+) T-cell response is associated with an upregulation of CD95 expression and a proapoptotic phenotype. Both can be blocked by adenoviral vaccination.

Analysis of the memory B cell response against glycoconjugate vaccines

The development of vaccines directed against polysaccharide capsules of S. pneumoniae, H. influenzae and N. meningitidis have been of great importance in preventing potentially fatal infections. Bacterial capsular polysaccharides are T-cell-independent antigens that induce specific antibody response characterized by IgM immunoglobulins, with a very low IgG class switched response and lack of capability of inducing a booster response. The inability of pure polysaccharides to induce sustained immune responses has required the development of vaccines containing polysaccharides conjugated to a carrier protein, with the aim to generate T cell help. It is clear that the immunogenicity of glycoconjugate vaccines can vary depending on different factors, e.g. chemical nature of the linked polysaccharide, carrier protein, age of the target population, adjuvant used. The present study analyzes the memory B cell (MBC) response to the polysaccharide and to the carrier protein following vaccination with a glycoconjugate vaccine for the prevention of Group B streptococcus (GBS) infection. Not much is known about the role of adjuvants in the development of immunological memory raised against GBS polysaccharides, as well as about the influence of having a pre-existing immunity against the carrier protein on the B cell response raised against the polysaccharide component of the vaccine. We demonstrate in the mouse model that adjuvants can increase the antibody and memory B cell response to the carrier protein and to the conjugated polysaccharide. We also demonstrate that a pre-existing immunity to the carrier protein favors the development of the antibody and memory B cell response to subsequent vaccinations with a glycoconjugate, even in absence of adjuvants. These data provide a useful insight for a better understanding of the mechanism of action of this class of vaccines and for designing the best vaccine that could result in a productive and long lasting memory response.

HLA class II mismatch alleles as targets of the alloreactive CD4 T-cell response

In allogeneic hematopoietic stem cell transplantation (allo-HSCT), alloreactive T lymphocytes of donor origin mediate the beneficial graft-versus-leukemia effect but also induce graft-versus-host disease (GvHD). Since human leukocyte antigens (HLA) mismatch alleles represent major targets of alloreactive T lymphocytes, patient and donor are usually matched for the class I molecules A, B, C, and for the class II molecules DRB1 and DQB1, in order do reduce the risk of GvHD. The HLA-DPB1 locus, however, is still ignored in donor selection. Interestingly, clinical studies have demonstrated that disparities at HLA-DQB1 alleles as well as distinct HLA DPB1 mismatch constellations do not adversely affect the outcome of allo-HSCT. It has also been shown that HLA class II is predominantly expressed on hematopoietic cells under non-inflammatory conditions. Therefore, this PhD thesis focused on the application of CD4 T cells in adoptive immunotherapy of leukemias.rnIn the first part of this thesis we developed a rapid screening approach to detect T-cell reactivity of donors to single HLA class II mismatch alleles. Allo-HLA reactivity was measured in naive, memory, and entire CD4 T cells isolated from PBMC of healthy donors by flow cytometric cell sorting according to expression of the differentiation markers CD45RA, CD45RO, CD62L, and CCR7. T-cell populations were defined by a single marker to facilitate translation into a clinical-grade allo-depletion procedure. Alloreactivity to single HLA-DR/-DQ mismatch alleles was analyzed in short-term mixed lymphocyte reactions (MLR) in vitro. As standard antigen-presenting cells, we used the HLA-deficient cell line K562 upon electroporation with single HLA-DR/-DQ allele mRNA. We observed in IFN-γ ELISpot assays that allo-HLA-reactivity preferentially derived from subsets enriched for naive compared to memory T cells in healthy donors, irrespective of the HLA mismatch allele. This separation was most efficient if CD62L (P=0.008) or CD45RA (P=0.011) were used as marker. Median numbers of allo-HLA-reactive effector cells were 3.5-fold and 16.6-fold lower in CD62Lneg and CD45RAneg memory CD4 T cells than in entire CD4 T cells, respectively. In allele-specific analysis, alloreactivity to single HLA-DR alleles clearly exceeded that to HLA-DQ alleles. In terms of alloproliferation no significant difference could be observed between individual CD4 T-cell subsets. rnThe second part of this thesis dealed with the generation of allo-HLA-DQ/-DP specific CD4 T cells. Naive CD45RApos CD4 T cells isolated from healthy donor PBMC by flow cytometric cell sorting were stimulated in MLR against single allo-HLA-DQ/-DP alleles transfected into autologous mature monocyte-derived dendritic cells by mRNA electroporation. Rapidly expanding HLA-DQ/-DP mismatch reactive T cells significantly recognized and cytolysed primary acute myeloid leukemia (AML) blasts, fibroblasts (FB) and keratinocytes (KC) in IFN-γ ELISpot and 51chromium release assays if the targets carried the HLA DQ/ DP allele used for T cell priming. While AML blasts were recognized independent of pre-incubating them with IFN-γ, recognition of FB and KC required IFN-γ pre treatment. We further investigated HLA class II expression on hematopoietic and non-hematopoietic cells by flow cytometry. HLA class II was not detected on primary FB, KC, and non-malignant kidney cells, but was expressed at significant levels on primary AML blasts and B-LCL. Up-regulation of HLA class II expression was observed on all cell types after pre-incubation with IFN-γ.rnIn summary, the novel K562-HLA based MLR approach revealed that naive-depleted CD4 T-cell subsets of healthy individuals contain decreased allo-HLA reactivity in vitro. We propose the application of CD45RAneg naive-depleted CD4 T cells as memory T cell therapy, which might be beneficial for HLA-mismatched patients at high-risk of GvHD and low-risk of leukemia relapse. Memory T cells might also provide important post-transplant immune functions against infectious agents. Additionally, the screening approach could be employed as test system to detect donors which have low risks for the emergence of GvHD after allo-HSCT. In the second part of this thesis we developed a protocol for the generation of allo-HLA-DQ/-DP specific CD4 T cell lines, which could be applied in situations in which patient and donor are matched in all HLA alleles but one HLA-DQ/-DP allele with low GvHD potential. These T cells showed lytic activity to leukemia cells while presumably sparing non-hematopoietic tissues under non-inflammatory conditions. Therefore, they might be advantageous for allo-HSCT patients with advanced stage AML after reduced-intensity conditioning and T-cell depletion for the replenishment of anti-leukemic reactivity if the risk for disease relapse is high. rn