MACS Separation Research Articles

Overcoming T cell–mediated rejection of bone marrow allografts by T-regulatory cells: Synergism with veto cells and rapamycin

Recently, we have shown that anti-third-party cytotoxic T lymphocytes (CTLs) depleted of alloreactivity against the host are endowed with marked veto activity and can facilitate bone marrow (BM) allografting without graft-versus-host disease. We also demonstrated synergism between rapamycin (RAPA) and the veto cells. CD4(+)CD25(+) T-regulatory (Treg) cells are suppressor cells that can enhance alloengraftment. We investigated whether donor Tregs would be synergistic with veto CTLs and RAPA in augmenting alloengraftment or, conversely, would suppress veto CTL effects. Lethally irradiated C3H mice were transplanted at day 2 after irradiation with Balb-nude BM. Graft rejection was induced by purified host-type T cells infused 1 day prior to BMT. The addition of Tregs led to moderate enhancement of engraftment. RAPA at different doses was synergistic with Tregs. The addition of veto CTLs to Tregs enabled reducing the effective RAPA dose fourfold. Combining all three agents was necessary to overcome rejection at low-dose RAPA. Chimerism analysis at 5 to 9 months revealed a significant presence of host-type cells coexisting with the predominant donor T cells, suggesting that tolerance had been attained. The synergistic effects between Tregs, veto CTLs, and RAPA offer an attractive approach for facilitating alloengraftment.

Immunomagnetic removal of cryo-damaged human spermatozoa

To estimate the dissipation of mitochondrial transmembrane potential (mTMP, DeltaPsim) and activation of sperm caspases (aCP) as signs of apoptosis in human spermatozoa during cryopreservation and to evaluate the efficiency of immunomagnetic cell separation (MACS) of these spermatozoa via annexin V-binding. The mTMP and aCP in fresh and cryopreserved spermatozoa were detected by fluorescence microscopy and by Western blots. The sperm suspensions were divided into two sperm fractions (with intact and deteriorated membranes) by magnetic cell separation (MiniMACS, Miltenyi Biotec, Bergisch Gladbach, Germany) in dependence on their binding to superparamagnetic annexin V-microbeads (AN-MB). The cryopreservation decreased the portion of spermatozoa with intact mTMP from 80.1 % +/- 7.2 % to 53.5 % +/- 13.1 % and increased the spermatozoa with activated pan-caspases (aCP) from 21.8 % +/- 2.6 % to 47.7 % +/- 5.8 % (n=10; mean +/- SEM; P <0.01). The activation of caspases 1, 3, 8, and 9 in the cryopreserved spermatozoa was confirmed by Western blots (n=22). MACS reduced significantly the percentage of cryopreserved spermatozoa with dissipated mTMP to 8.1 +/- 3.9 (P <0.01) and also those with aCP to 9.3 % +/- 2.2 %. Western blot analyses confirmed the increase of the activated caspase3, 9, and 8 in the AN-MB-positive fraction (P <0.05) compared with the AN-MB-negative fraction. The MACS separation effect was confirmed by anti-annexin V-antibodies. There was no significant influence of the separation column and the magnetic field on the sperm functions. The cryopreservation impaired the mTMP and enhanced the activation status of caspases in human spermatozoa. The immunomagnetic sperm separation via binding of AN-MB could deplete low quality spermatozoa from cryopreserved semen samples.

Repeated Topical Challenge with Chemical Antigen Elicits Sustained Dermatitis in NC/Nga Mice in Specific-Pathogen-Free Condition

NC/Nga mice are known to develop skin lesions resembling to atopic dermatitis (AD) in conventional but not in specific-pathogen-free (SPF) condition. An epicutaneous application of 2,4-dinitrofluorobenzene (DNFB) increased skin thickness in C3H as well as NC/Nga mice in SPF environment, and the response was enlarged by repeating the challenge at weekly intervals. Although the skin reaction in C3H mice was ameliorated when the challenge was discontinued after the fifth application, the reaction in NC/Nga mice was sustained at least for 3 wk. Analyses of cytokine production by CD4+ cells from the draining lymph node proximal to the lesions revealed that, unlike C3H mice, NC/Nga mice fail to induce T helper 2 (Th2) cytokine interleukin-4 (IL-4), whereas the level of Th1 cytokine interferon-gamma in NC/Nga mice is equivalent to that of C3H mice. In addition, NC/Nga mice highly expressed IL-12, a cytokine-preventing formation of Th2 response, whereas C3H mice did not. Administration of anti-IL-12 antibody reduced duration of dermatitis in DNFB-treated NC/Nga mice. Taken together, our data suggest that IL-12 plays a role in the persistent skin reaction in NC/Nga mice. The action of IL-12 might be mediated by the decrease in IL-4 production.

Murine respiratory tract dendritic cells: isolation, phenotyping and functional studies

Respiratory tract dendritic cells (RTDC) form a contiguous subepithelial network within the nasorespiratory tract bridging innate and acquired immunity and have been implicated in nasal mucosal tolerance induction. Discrepancies exist between isolation techniques with respect to phenotype and function of RTDC. Therefore, the aim of this study was to modify previous methods to provide a consistent isolation method whilst maintaining good cell viability and enriched cell numbers so as to facilitate further phenotype and functional studies of murine RTDC. RTDCs isolated by enzyme digestion, Percoll density gradient centrifugation and overnight GM-CSF culture followed by MACS separation retain an archetypical immature dendritic cell phenotype, characterised by MHCII low CD40 neg CD86 neg CD80 neg CD11c low cell surface expression. Splenic-derived DC (SDC) isolated conformed to a day 1 in vitro phenotype; MHCII low CD40 neg CD86 low CD80 neg CD11c low but can further mature phenotypically in vitro. Both RTDC and SDC processed and presented antigen efficiently to T cells in vitro. Using such modified isolation procedures for RTDCs, we have developed a consistent method of RTDC enrichment, which maintains the immature phenotype and functional antigen presenting capability.

Essential Role of p38 Mitogen-activated Protein Kinase in Contact Hypersensitivity

The present study was designed to elucidate the role of p38 mitogen-activated protein kinase (p38) in the pathogenesis of inflammation, using a mouse contact hypersensitivity (CHS) model induced by 2,4-dinitro-1-fluorobenzene (DNFB). Ear swelling was induced by challenge with DNFB, accompanied by infiltration of mononuclear cells, neutrophils, and eosinophils and a marked increase in mRNA levels of cytokines such as interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-5, IL-1beta, IL-18, and tumor necrosis factor-alpha in the challenged ear skin. Both ear swelling and the number of infiltrated cells in DNFB-challenged ear skin were significantly inhibited by treatment with SB202190, a p38 inhibitor. Furthermore, the DNFB-induced expression of all cytokines except IL-4 was significantly inhibited by treatment with SB202190. Ribonuclease protection assay revealed that the mRNA levels of chemokines such as IP-10 and MCP-1 in ear skin were markedly increased at 24 h after challenge with DNFB. The induction of these chemokines was significantly inhibited by treatment with SB202190. In p38alpha +/- mice, both ear swelling and infiltration of cells induced by DNFB were reduced compared with those in wild-type mice. However, induction of cytokines by DNFB was also observed in p38alpha +/- mice, although the induction of IFN-gamma, IL-5, and IL-18 was typically reduced compared with that in wild-type mice. Challenge with DNFB slightly induced IP-10 and MCP-1 mRNA in p38alpha +/- mice, with weaker signals than those in SB202190-treated wild-type mice. These results suggest that p38 plays a key role in CHS and is an important target for the treatment of CHS.

A simple and rapid magnetic bead separation technique for the isolation of tetramer-positive virus-specific CD8 T cells

A simple and rapid magnetic bead separation technique for the isolation of tetramer-positive virus-specific CD8 T cells

Immature B lymphocytes from adult bone marrow exhibit a selective defect in induced hyperexpression of major histocompatibility complex class II and fail to show B7.2 induction.

Mature B lymphocytes respond to antigen receptor ligation by phenotypic changes, including up-regulation of major histocompatibility complex class II molecules and expression of B7.2, which are required for initiating and sustaining a productive interaction with T helper cells. We have previously demonstrated that neonatal B cells fail to show a similar up-regulation of class II and B7.2 expression following B-cell receptor (BCR) ligation, although these responses could be induced by other stimuli. Here we demonstrate that immature B cells from adult bone marrow exhibit even more profound defects in these responses, as they fail to up-regulate class II in response to either BCR ligation or interleukin-4. Moreover, bone marrow-derived, immature B cells could not be induced to express B7.2 either by receptor cross-linking or by lipopolysaccharide. These differences in the inducible expression of class II and B7.2 appear to be intrinsic to the B cells, as they were retained in purified populations of B-lineage cells and could not be induced in mature B cells by co-culture with bone marrow cells. Furthermore, short-term culture of bone marrow permitted B-cell maturation, which was accompanied by acquisition of responsiveness to the same stimuli as mature, splenic B cells. The inability of immature B cells to show these responses provides a molecular explanation for their reported deficiency in interacting with T cells. Failure of immature B cells to inducibly express B7.2 may also be important for the establishment of self tolerance in the B-cell compartment.

Development of rat CD45+ 13-day-old fetal liver cells in SCID mouse fetal thymic organ cultures.

A phenotypic analysis of the lympho-hemopoietic cells which occur in the liver of 13-day-old fetal rats was achieved by flow cytometry in an attempt to further characterize the rat lymphoid progenitor cells. A small fraction of rat 13-day-old fetal liver (r13FL) cells, which weakly expressed the leukocyte common antigen CD45, constituted a homogeneous Thy-1(hi), CD71(-), CD44(+), MHC class I+, CD43(+) cell subpopulation negative for CD45RC, CD3, TCRalphabeta, TCRgammadelta, CD2, CD5, CD4, CD8, CD25, CD28, NKR-P1a and sIg. On the contrary, the CD45(-) cells were a heterogeneous cell subset which expressed Thy-1, CD71 and CD44 at distinct levels. After MACS separation, the CD45(+) r13FL cells, but not the CD45(-) cell subset, in vitro repopulated 14-day-old SCID mouse fetal thymic lobes providing rat T cells, both TCRalphabeta and TCRgammadelta, NK cells, and thymic dendritic cells but not B lymphocytes. Interestingly, NKR-P1a(lo) TCRalphabeta+ or TCRgammadelta+ cells developed in the xenogeneic cultures, and a rare CD4(+)CD8(+) double-positive subpopulation among the TCRgammadelta-expressing cells accumulated in the oldest cultures. These results are discussed from the double perspective of the nature of the precursor cells which colonize the fetal thymus and the relevance of the xenogeneic SCID mouse fetal thymic microenvironment for supporting rat lymphopoiesis.