Macrophages but not B cells from aged mice are defective in stimulating autoreactive T cells in vitro

In the present study the effect of aging on the capacity of Ia+ cells to stimulate autoreactive T cells in the syngeneic mixed lymphocyte reaction (SMLR) was investigated. Using young CD4+ T cells as responders, it was observed that unseparated whole spleen cells from aged mice had normal stimulatory activity comparable to that of young spleen cells. Interestingly, however, when purified splenic adherent cells (SAC) enriched for macrophages or splenic B cells were used as stimulators, aged SAC but not aged B cells were found to be defective in stimulating autoreactive T cells. This defect in aged SAC was not due to decreased expression of Ia antigens since the percentage of Ia+ SAC and density of Ia antigen expression was similar in both young and old mice. Also, the B cells from aged mice expressed normal levels of Ia antigens. Aged SAC, when mixed with young SAC could also actively suppress the normal SMLR. However, this suppression was not due to increased prostaglandin production but was found to be associated with interleukin-1 (IL-1) regulation, inasmuch as addition of exogenous IL-1 could completely reconstitute the defective stimulatory activity of aged SAC and also abolished the suppressor activity of the SAC. Aged mice also demonstrated an intrinsic defect in the CD4+ T cells responding in the SMLR. Together, our studies on the SMLR demonstrate an age-related defect in responder autoreactive T cells and in stimulator splenic macrophages but not in the stimulatory activity of B cells.     

The long-term antibody response of New Zealand Black mice to sheep red blood cells

Long-term IgM and IgG responses to sheep red blood cells (SRBC) were determined in New Zealand Black (NZB) mice, using the plaque technique. As compared with other strains a prolonged spleen plaque-forming cell (PFC) response was found in young and old NZB mice. This phenomenon was found in the primary as well as in the secondary response and concerned IgM as well as IgG production. These observations are discussed in relation to the production of IgG antibodies of a low avidity.     

Homing of lymphocytes into islets of Langerhans in prediabetic non-obese diabetic mice is not restricted to autoreactive T cells

Non-obese diabetic mice spontaneously develop a type 1 diabetes.The entry of leukocytes in the islets of Langerhans was studiedin untreated and in irradiated mice. FITC-labeled cells fromspleen, lymph nodes or bone marrow of healthy or diabetic donorsdid home to the inflamed islets of unmanipulated recipients.B and T cells migrated equally well, whereas rare neutrophilsentered the islets. Lymphocyte homing was blocked by anti-L-selectinand anti-4 integrin antibodies.Insulitis transfer experimentsusing mice congenic at the Thy-1 locus showed that anti-4 integrintreatment totally inhibited the migration of donor type T cellsin the islets, whereas anti-L-selectin only had an early andtransient effect. The expression of vascular addressins in theislets was linked to the presence of mononuclear cells. Thus,in the developing islet infiltrate, the entry of cells appearscontinuous and restricted to lymphocytes, whether autoreactiveor not, and involves the L-selectin. This mechanism rather promotesthe migration of naive-type cells. Conversely,during the adoptivetransfer of insulitis the entry of L-selectin^– diabetogenicT cells is highly favored, to the detriment of L-selectin⁺ naivetype cells.     

Antibody binding to a collagen type-II epitope gives rise to an inhibitory peptide for autoreactive T cells.

It is well documented that antigen recognition by T cells requires small peptides which are generated by protein cleavage in antigen-presenting cells. These peptides have to associate with major histocompatibility complex (MHC) molecules in order to be recognized. An inhibitory peptide may bind to the same site of the MHC-encoded protein but is not recognized by the T cell. Here we describe a stimulatory and an inhibitory peptide sequence within human collagen type II (CII) as defined by means of the same autoreactive human T cell clone. Most interestingly, the inhibitory peptide is not generated by regular processing in peripheral blood mononuclear cells but only in the presence of an antibody that binds to the same domain and thereby seems to protect the inhibitory sequence. This finding may indicate that certain autoantibodies have the potential to block autoreactive T cells with specificity for a distinct epitope on the same antigen.     

New Zealand black mice are immunologically resistant to high-dose, but not low-dose Leishmania mexicana infection.

The course of infection following s.c. inoculation of a wide dose range of L. mexicana stationary-phase promastigotes (SPP) was examined in sexually mature and immature NZB mice of both sexes. Infection with a high dose (greater than 10(7) SPP) was able to induce a protective in vivo response, which could be adoptively transferred with parasite-immune T cells, into naive, syngeneic recipients. In contrast, s.c. infection with a low dose (less than 10(7) SPP) induced non-healing lesions; disease susceptibility could also be transferred into naive animals with T cells from non-immune donors. When the ability to mount a delayed-type hypersensitivity (DTH) reaction was tested in these two groups, the high-parasite dose group gave a significantly higher response. The in vivo protection and high DTH response were reflected in the ability of cells derived from the high-dose resistant (but not low-dose susceptible) mice to mount an antigen-specific T cell response in vitro. The possible immunological effector mechanisms underlying high-dose resistance and low-dose susceptibility are discussed.     

T cells are required for host protection against Brugia malayi but need not produce or respond to interleukin-4

T cells are known to be required for host protection in mouse models of Brugia malayi infection. Several independent studies in murine models have also highlighted the rapid induction of Th2-like responses after infection with B. malayi or B. pahangi. Previous data from our laboratory have described a significant increase in permissiveness in the absence of interleukin-4 (IL-4), the "prototypical" Th2 cytokine, involved in both the induction and maintenance of Th2 responses. These observations led to our hypothesis that T cells involved in murine host protection would respond to IL-4 signaling and differentiate into cells of the "type 2" phenotype. As such, these cells would presumably also act as major sources of IL-4. To investigate these hypotheses, we performed several adoptive transfers in which we controlled the cell population(s) able to produce or respond to IL-4. We show here that, in contrast to our original hypotheses, IL-4 production and IL-4 receptor expression by T cells are both dispensable for T-cell-mediated host protection. Instead, our data imply that T cells may be required for eosinophil accumulation at the site of infection.     

The majority of HLA-DR3 alloreactive T cells is peptide specific, but does not recognize known DR3-bound sequences

Abstract: Rejection of transplants is frequently caused by activation of alloreactive T cells that recognize HLA/peptide differences between patient and graft. This T-cell response can be directed towards the HLA molecule, the HLA-bound peptide or towards a combination. More insight in the involvement of peptides in this process may help to find ways to avoid rejection using for example antagonist peptides. In recent years many naturally processed HLA-bound peptides have been identified. This raises the question of whether these, presumably abundant, peptides are involved in class ri-specific allorecognition. To investigate this, we first determined the proportion of peptide-specific alloreactive T cells in the alloresponse against HLA-DR3. For this purpose we have tested a panel of DR3-specific alloreactive T-cell clones against a DM-mutant (i.e. peptide loading deficient) cell line. We found that 59 out of 64 alloreactive T-cell clones were dependent upon the presence of DM for an optimal response. However, only 2 DM-dependent T-cell clones recognize known peptide sequences. Thus we conclude that most DR3-specific alloreactive T-cell clones are peptide specific and that the currently known DR3-bound peptides are not the main target for allorecognitioa Finally, we identified 4 T-cell clones that recognized the DM-mutant better than the wild-type cell line. The response against the wild-type cell line could not be restored with invariant chain derived peptides (CLIP). This provides additional evidence that DM can negatively select self-peptides other than CLIP, which can result in selection against peptides involved in allorecognition.     

Development of autoreactive L3T4+ T cells from double-negative (L3T4-/Ly-2-) Thy-1+ spleen cells of normal mice

Thy-1+/L3T4-/Ly-2- spleen cells were purified from normal C57BL/6 (B6) and C,B-17 mice. Cells within this subset expressed the T cell receptor (TcR) for antigen: the majority of cells in this subset were CD3+; a fraction of the cells was stained with the monoclonal antibody (mAb) F23.1; and the TcR molecule was immunoprecipitable with mAb F23.1 from cells within this subset. In limiting dilution analyses, about 1/30 cells within this subset were growth inducible in vitro by stimulation with phorbol myristate acetate (PMA) plus ionomycin; conditioned media containing interleukin (IL) 1, IL2, IL3 or IL4 activity neither triggered nor promoted in vitro growth of these cells. The in vitro generated T cells displayed the Thy-1+/L3T4+/Ly-2- surface phenotype and were self-reactive, i.e., proliferated preferentially in response to syngeneic stimulator cells, and secreted IL2 and IL3 only in response to syngeneic but not allogeneic stimulator cells. The proliferative response of these cells to syngeneic stimulator cells was blocked by anti-self Ia mAb. This autoreactive helper T cell subset was not inducible in purified Thy-1+ spleen cell subsets from athymic nude mice or scid mice. Autoreactive helper T cells did not express detectable levels of the IL2 receptor (IL2R), and their proliferative response was not blocked by anti-IL2R mAb. From PMA plus ionomycin-stimulated double-negative Thy-1+ spleen cells, 14 T cell clones were established in long-term culture which displayed the CD3+CD4+CD8- surface phenotype and were self-reactive.     

AMPD3‐deficient mice exhibit increased erythrocyte ATP levels but anemia not improved due to PK deficiency

AMP deaminase (AMPD) catalyzes AMP to IMP and plays an important role in energy charge and nucleotide metabolism. Human AMPD3 deficiency is a type of erythrocyte‐specific enzyme deficiency found in individuals without clinical symptoms, although an increased level of ATP in erythrocytes has been reported. To better understand the physiological and pathological roles of AMPD3 deficiency, we established a line of AMPD3‐deficient [A3(?/?)] mice. No AMPD activity and a high level of ATP were observed in erythrocytes of these mice, similar to human RBC‐AMPD3 deficiency, while other characteristics were unremarkable. Next, we created AMPD3 and pyruvate kinase (PK) double‐deficient [PKA(?/?,?/?)] mice by mating A3(?/?) mice with CBA‐Pk‐1slc/Pk‐1slc mice [PK(?/?)], a spontaneous PK‐deficient strain showing hemolytic anemia. In PKA(?/?,?/?) mice, the level of ATP in red blood cells was increased 1.5 times as compared to PK(?/?) mice, although hemolytic anemia in those animals was not improved. In addition, we observed osmotic fragility of erythrocytes in A3(?/?) mice under fasting conditions. In contrast, the ATP level in erythrocytes was elevated in A3(?/?) mice as compared to the control. In conclusion, AMPD3 deficiency increases the level of ATP in erythrocytes, but does not improve anemia due to PK deficiency and leads to erythrocyte dysfunction.     

Human mesenchymal stem cells respond to native but not oxidized damage associated molecular pattern molecules from necrotic (tumor) material

Necrosis is a characteristic feature of advanced solid tumors. Released necrotic factors, also referred to as damage associated molecular patterns (DAMPs), are known to critically impact the tumor microenvironment by enhancing angiogenesis or influencing the immune response. We have recently shown that DAMPs can act as chemoattractants and activators of granulocytes. We demonstrate that necrotic material from both normal and tumor cells promotes proliferation and trafficking of human mesenchymal stem cells (MSCs). We characterize the protein high mobility group box 1 (HMGB1) as a crucial member of DAMPs within necrotic material. In addition, we show that DAMPs interfere with expression of indoleamine 2, 3-dioxygenase (IDO) in MSCs. The biological activity of necrotic material toward MSCs is abolished once these DAMPs are oxidized. MSCs found within tumor tissue can act as immunoregulatory cells and are able to promote tumor metastasis, thus playing a crucial role within the tumor microenvironment. Here, we reveal DAMPs to be crucial factors in the setting of MSC biology within the tumor microenvironment. The tumor microenvironment is characterized by reducing and hypoxic conditions that protect DAMPs from oxidation. Based on our results, oxidizing conditions should be considered for therapeutic approaches that target the tumor microenvironment.     

CD8 T cells from major histocompatibility complex class II-deficient mice respond vigorously to class II molecules in a primary mixed lymphocyte reaction

Mature CD4⁺ and CD8⁺ T cells are restricted by major histocompatibility complex (MHC) class II and class I molecules, respectively. In a primary mixed lymphocyte reaction (MLR), CD8⁺ T cells from C57BL/6 (B6) mice can respond to allo-class I molecules, but not allo-class II molecules. However, a significant fraction of CD8⁺ T cells from C57BL/6 class II-deficient (B6Aα^?) mice violate this rule by responding vigorously in a MLR to class II molecules. The frequency of responding cells is ～ 50 % of that of B6 CD8⁺ T cells responding to B6bm1 allo-class I molecules. This response requires neither appropriate co-receptor, i.e. CD4, nor exogenous lymphokines, indicating that interactions between the T cell receptors (TCR) and class II molecules are remarkably efficient. Since these CD8⁺ T cells are positively selected by class I molecules in the thymus of class II-deficient mice, these CD8⁺ T cells should interact with both classes of MHC molecules. The absence of thymic negative selection by class II molecules may result in the production of these CD8⁺ T cells. The data imply that a substantial fraction of CD4⁺CD8⁺ double-positive thymocytes in wild-type mice interacts with both classes of MHC molecules prior to thymic selection.     

Differential IL-12 responsiveness of T cells but not of NK cells from tumor-bearing mice in IL-12-responsive versus -unresponsive tumor models

While IL-12 administration induces tumor regression through stimulating T cells in tumor-bearing mice, this IL-12 effect is observed in some but not all tumor models. The present study aimed to compare IL-12 responsiveness of T cells from tumor-bearing mice in IL-12-responsive (CSA1M and OV-HM) and -unresponsive (Meth A) tumor models. Tumor regression in IL-12-responsive tumor models required the participation of T cells, but not of NK1.1(+) cells. Because a NK1.1(+) cell population was the major producer of IFN-gamma, comparable levels of IFN-gamma production were induced in IL-12-responsive and -unresponsive tumor-bearing mice. This indicates that the amount of IFN-gamma produced in tumor-bearing individuals does not correlate with the anti-tumor efficacy of IL-12. In contrast, IL-12 responsiveness of T cells differed between the responsive and unresponsive models: purified T cells from CSA1M/OV-HM-bearing or Meth A-bearing mice exhibited high or low IL-12 responsiveness respectively, when evaluated by the amounts of IFN-gamma produced in response to IL-12. T cells from CSA1M- or OV-HM-bearing but not from Meth A-bearing mice exhibited enhanced levels of mRNA for the IL-12 receptor (IL-12R). These results indicate that a fundamental difference exists in IL-12 responsiveness of T cells between IL-12-responsive and -unresponsive tumor models, and that such a difference is associated with the expression of IL-12R on T cells.     

Autistic effector T cells in mice with a point mutation in the LAT adaptor fail to respond to Listeria monocytogenes infection

The adaptor protein linker for activation of T cells (LAT) is an important transducer of extracellular T cell stimuli. In mice with a point mutation in LAT (LatY136F), TCR signaling is substantially compromised and LatY136F T cells are unresponsive to CD3 cross-linking in vitro. Nevertheless, LatY136F mice develop a polyclonal lymphoproliferation of CD4(+) T cells, which display a T(h)2-polarized effector phenotype. In this study, LatY136F mice were infected with the intracellular bacterium Listeria monocytogenes and the antigen-specific responses of T cells were determined. Both CD4(+) and CD8(+) LatY136F T cells were unresponsive to L. monocytogenes infection. In contrast, when CD4(+) T cells from wild-type mice were adoptively transferred into LatY136F hosts, they responded normally to L. monocytogenes, indicating that the LatY136F milieu permits T(h)1 responses. Furthermore, we analyzed whether the infection would influence the capacity of LatY136F CD4(+) T cells to produce IL-4 and IFN-gamma. While L. monocytogenes infection results in T(h)1-type T cell responses in wild-type animals, we found that it did not shift the strong T(h)2 polarization of LatY136F T cells towards a T(h)1 pattern. In conclusion, our results suggest that the activation and T(h)2 polarization of the LatY136F CD4(+) T cells is not influenced by infection with an intracellular pathogen known to induce robust T(h)1 responses, and is thus likely driven by T cell intrinsic mechanisms.     

Cytokine production of CD8+ immune T cells but not of CD4+ T cells from Toxoplasma gondii-infected mice is polarized to a type 1 response following stimulation with tachyzoite-infected macrophages.

To examine whether cytokine production of CD4(+)immune T cells and CD8(+)immune T cells in Toxoplasma gondii-infected mice differ in their responses to infected cells and to soluble antigens of the parasite, we compared the production of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-10 by the immune T cell populations following in vitro stimulation with tachyzoite-infected macrophages and tachyzoite lysate antigens (TLA). Both CD4(+)and CD8(+)immune T cells produced large amounts of IFN-gamma in response to either infected macrophages or TLA, but the CD4(+)T cells produced greater amounts of the cytokine than did the CD8(+)T cells with both stimulations. Both T cell populations also produced IL-2 after stimulation with infected macrophages, whereas only CD4(+)T cells did when stimulated with TLA. CD4(+)immune T cells also produced large amounts of IL-4 and IL-10 after stimulation with infected macrophages, but CD8(+)T cells did not. These results indicate that CD4(+)immune T cells produce IFN-gamma, IL-2, IL-4, and IL-10 in response to infected macrophages, whereas CD8(+)immune T cells produce predominantly IFN-gamma and IL-2. Since IL-4 and IL-10 could suppress IFN-gamma-mediated protective mechanisms against the parasite, the production of these cytokines by CD4(+)immune T cells in response to infected cells could negatively affect their protective activity in vivo.     

Staphylococcal protein A bound to Sepharose 4B is mitogenic for T cells but not B cells from rabbit tissues

The mitogenicity of protein A from Staphylococcus aureus bound to Sepharose 4B (SpA-S) was tested against mononuclear cells from normal rabbit spleens and Peyer's patches after using Sephadex G-10 adherence chromatography to deplete macrophages and Sephadex G-200 anti-rabbit F(ab')2 immunoabsorbent columns to obtain B-enriched lymphocytes. Macrophage-depleted unseparated lymphocytes and the B-cell-depleted (T-cell-enriched) fractions from both tissues consistently showed good mitogenic responses to SpA-S. In contrast, the B-cell-enriched populations from either tissue did not respond to SpA-S. Supplementary experiments employing glass adherence and T-cell autorosetting did not support the possibility that the more efficient immunoabsorbent method of B-cell isolation had in some way caused the unresponsiveness to SpA-S. These results indicate that SpA-S is mitogenic for rabbit tissue T cells but will not serve as a T-independent mitogen for rabbit tissue B cells.     

Renal dendritic cells adopt a pro-inflammatory phenotype in obstructive uropathy to activate T cells but do not directly contribute to fibrosis

Unilateral ureteral obstruction (UUO) is a well-characterized murine model of renal inflammation leading to fibrosis. Renal dendritic cells (DCs) constitute a significant portion of kidney leukocytes and may participate in local inflammation and have critical roles in antigen presentation. The heterogeneity in renal DC populations and surface marker overlap with monocytes/macrophages has made studying renal DCs difficult. These studies used CD11c-promoter driven reporter/depletion mice to study DCs in vivo. Studying early local inflammatory events (day 3 of UUO), in vivo multiphoton imaging of the intact kidney of CD11c reporter mice revealed more dendrite extensions and increased activity of renal DCs in real time. Phenotypic analysis suggested resident DC maturation in obstructed kidneys with increased CD11b and less F4/80 expressed. CD11b(hi) Gr-1(+) inflammatory DCs were also present in obstructed kidneys. T-cell receptor transgenic mice revealed enhanced antigen-presenting capacity of renal DCs after UUO, with increased antigen-specific T-cell proliferation in vivo and ex vivo. However, conditional DC ablation at days 0, 2, or 4 did not attenuate fibrosis or apoptosis 7 days after UUO, and depletion at 7 days did not alter outcomes at day 14. Therefore, after UUO, renal DCs exhibit inflammatory morphological and functional characteristics and are more effective antigen-presenting cells, but they do not directly contribute to tubulointerstitial damage and fibrosis.