Presence Of Alpha Beta Research Articles

Extensive expansion of primary human gamma delta T cells generates cytotoxic effector memory cells that can be labeled with Feraheme for cellular MRI.

Gamma delta T cells (GDTc) comprise a small subset of cytolytic T cells shown to kill malignant cells in vitro and in vivo. We have developed a novel protocol to expand GDTc from human blood whereby GDTc were initially expanded in the presence of alpha beta T cells (ABTc) that were then depleted prior to use. We achieved clinically relevant expansions of up to 18,485-fold total GDTc, with 18,849-fold expansion of the Vδ1 GDTc subset over 21 days. ABTc depletion yielded 88.1 ± 4.2 % GDTc purity, and GDTc continued to expand after separation. Immunophenotyping revealed that expanded GDTc were mostly CD27-CD45RA- and CD27-CD45RA+ effector memory cells. GDTc cytotoxicity against PC-3M prostate cancer, U87 glioblastoma and EM-2 leukemia cells was confirmed. Both expanded Vδ1 and Vδ2 GDTc were cytotoxic to PC-3M in a T cell antigen receptor- and CD18-dependent manner. We are the first to label GDTc with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles for cellular MRI. Using protamine sulfate and magnetofection, we achieved up to 40 % labeling with clinically approved Feraheme (Ferumoxytol), as determined by enumeration of Perls' Prussian blue-stained cytospins. Electron microscopy at 2,800× magnification verified the presence of internalized clusters of iron oxide; however, high iron uptake correlated negatively with cell viability. We found improved USPIO uptake later in culture. MRI of GDTc in agarose phantoms was performed at 3 Tesla. The signal-to-noise ratios for unlabeled and labeled cells were 56 and 21, respectively. Thus, Feraheme-labeled GDTc could be readily detected in vitro via MRI.

Pathogenic role for skin macrophages in a mouse model of keratinocyte-induced psoriasis-like skin inflammation

Psoriasis is a common skin disease, the pathogenesis of which has not yet been resolved. In mice, epidermis-specific deletion of inhibitor of NF-kappaB (IkappaB) kinase 2 (IKK2) results in a skin phenotype that mimics human psoriasis in several aspects. Like psoriasis, this skin disease shows pronounced improvement when mice are treated with a TNF-neutralizing agent. We have found previously that this phenotype does not depend on the presence of alphabeta T lymphocytes. In order to evaluate contributions of other immune cell populations to the skin disease, we selectively eliminated macrophages and granulocytes from the skin of mice with epidermis-specific deletion of IKK2 (K14-Cre-IKK2fl/fl mice). Elimination of skin macrophages by subcutaneous injection of clodronate liposomes was accompanied by inhibition of granulocyte migration into the skin and resulted in a dramatic attenuation of psoriasis-like skin changes. The hyperproliferative, inflammatory skin disease in K14-Cre-IKK2fl/fl mice was a direct consequence of the presence of macrophages in the skin, as targeted deletion of CD18, which prevented accumulation of granulocytes but not macrophages, did not lead to major changes in the phenotype. Targeted deletion of the receptor for IFN-gamma revealed that the pathogenesis of the skin disease does not depend on classical IFN-gamma-mediated macrophage activation. Our results demonstrate that in mice epidermal keratinocytes can initiate a hyperproliferative, inflammatory, IFN-gamma-independent, psoriasis-like skin disease whose development requires essential contributions from skin macrophages but not from granulocytes or alphabeta T lymphocytes.

Immune or normal gamma delta T cells that assist alpha beta T cells in elicitation of contact sensitivity preferentially use V gamma 5 and V delta 4 variable region gene segments.

In the current study, we confirmed previous findings suggesting that gamma delta T cells were involved in the successful adoptive cell transfer of contact sensitivity (CS) by alpha beta CS-effector T cells. In this study, we used hamster anti-mouse gamma delta-TCR mAb treatment of CS-effector T cells, followed by enrichment and removal of the gamma delta T cells with goat anti-hamster Ig-linked magnetic beads, or by addition of hemolytic rabbit C. This removal of gamma delta T cells abrogated adoptive cell transfers of CS, despite the presence of alpha beta T cells that are known to mediate CS. FACS analysis documented enrichment of gamma delta T cells rising from 1 to 2% of the starting cells, to 60 to 95% of the magnetic bead adherent cells. Adoptive cell transfer of CS was reconstituted by adding back to the alpha beta cells, highly enriched gamma delta cells attached to anti-gamma delta-TCR magnetic beads. Not only were gamma delta-enriched T cells from sensitized mice able to assist immune CS-effector alpha beta T cells, but gamma delta T cells from normal nonimmune mice also had CS-assisting activity, and furthermore, neither were MHC-restricted in this function. Thus, CS-assisting gamma delta T cells were present endogenously in normal mice without prior immunization, and acted without Ag specificity and without MHC restriction, to assist CS-effector alpha beta T cells. Similar studies, with hamster mAbs specific for V gamma and V delta portions of gamma delta-TCR, demonstrated that the gamma delta T cells that assisted the CS-effector alpha beta T cells preferentially expressed V gamma 5 and V delta 4 in their TCR. PCR analysis on extracted mRNA showed that V gamma 5 and V delta 4 gene segments indeed were rearranged and expressed in the sensitized and normal lymph nodes; and one-and two-color FACS analysis of magnetic bead-fractionated cells suggested that V gamma 5 and V delta 4 were expressed on the same T cells. In summary, these results demonstrated that V gamma 5+, V delta 4+, gamma delta T cells were needed to assist alpha beta effector T cells in the adoptive cell transfer of CS.

Expansion of gamma delta+ T cells in BALB/c mice infected with Leishmania major is dependent upon Th2-type CD4+ T cells

T cells belong to either the alpha beta+ or gamma delta+ lineage as defined by their antigen receptor. Although both T-cell subsets have been shown to be involved in the immune response to the parasite Leishmania major, very little is known about possible interactions between these two populations. In this study, using a mouse model of infection with L. major, we showed that expansion of a subset of gamma delta+ T cells in vivo is dependent upon the presence of alpha beta+ CD4+ T cells. Moreover, this effect appears to be mediated via the secretion of lymphokines by CD4+ cells with a T-helper 2 (Th2) functional phenotype. Results showing that activation of Th2-type cells in mice treated with anti-immunoglobulin D antibodies or infected with Nippostrongylus brasiliensis also results in gamma delta+ T-cell expansion suggest that this effect of the Th2-type CD4+ cells is a general phenomenon not restricted to infection with L. major.