CXCR5 + CD8 + T Cell-mediated Suppression of Humoral Alloimmunity and AMR in Mice Is Optimized With mTOR and Impaired With Calcineurin Inhibition.

BACKGROUND: Adoptive cellular therapy (ACT) with antibody-suppressor CXCR5 + CD8 + T cells (CD8 + T Ab-supp ) inhibits alloantibody production, antibody-mediated rejection (AMR), and prolongs graft survival in multiple transplant mouse models. However, it is not known how conventional immunosuppressive agents impact the efficacy of CD8 + T Ab-supp ACT. METHODS: We investigated the efficacy of CD8 + T Ab-supp cell ACT when combined with calcineurin inhibitor (CNi) or mammalian target of rapamycin inhibitor (mTORi) in a murine model of kidney transplant. RESULTS: ACT-mediated decrease in germinal center B cells, posttransplant alloantibody titer, and amelioration of AMR in high alloantibody-producing CCR5 knockout kidney transplant recipients were impaired when ACT was combined with CNi and enhanced when combined with mTORi. CNi (but not mTORi) reduced ACT-mediated in vivo cytotoxicity of IgG + B cells and was associated with increased quantity of germinal center B cells. Neither CNi nor mTORi treatment impacted the expression of cytotoxic effector molecules (FasL, Lamp1, perforin, granzyme B) by CD8 + T Ab-supp after ACT. Concurrent treatment with CNi (but not mTORi) reduced in vivo proliferation of CD8 + T Ab-supp after ACT. The increase in quantity of splenic CD44 + CXCR5 + CD8 + T cells that occurs after ACT was reduced by concurrent treatment with CNi but not by concurrent treatment with mTORi (dose-dependent). CONCLUSIONS: Impaired efficacy of ACT by CNi is attributed to reduced persistence and/or expansion of CD8 + T Ab-supp cells after ACT. In contrast, concurrent immunosuppression with mTORi preserves CD8 + T Ab-supp cells quantity, in vivo proliferation, and in vivo cytotoxic effector function after ACT and enhances suppression of humoral alloimmunity and AMR.

Antibody-suppressor CXCR5+ CD8+ T cellular therapy ameliorates antibody-mediated rejection following kidney transplant in CCR5 KO mice.

CCR5 KO kidney transplant (KTx) recipients are extraordinarily high alloantibody producers and develop pathology that mimics human antibody-mediated rejection (AMR). C57BL/6 and CCR5 KO mice (H-2b ) were transplanted with A/J kidneys (H-2a ); select cohorts received adoptive cell therapy (ACT) with alloprimed CXCR5+ CD8+ T cells (or control cells) on day 5 after KTx. ACT efficacy was evaluated by measuring posttransplant alloantibody, pathology, and allograft survival. Recipients were assessed for the quantity of CXCR5+ CD8+ T cells and CD8-mediated cytotoxicity to alloprimed IgG+ B cells. Alloantibody titer in CCR5 KO recipients was four-fold higher than in C57BL/6 recipients. The proportion of alloprimed CXCR5+ CD8+ T cells 7 days after KTx in peripheral blood, lymph node, and spleen was substantially lower in CCR5 KO compared to C57BL/6 recipients. In vivo cytotoxicity towards alloprimed IgG+ B cells was also reduced six-fold in CCR5 KO recipients. ACT with alloprimed CXCR5+ CD8+ T cells (but not alloprimed CXCR5- CD8+ or third-party primed CXCR5+ CD8+ T cells) substantially reduced alloantibody titer, ameliorated AMR pathology, and prolonged allograft survival. These results indicate that a deficiency in quantity and function of alloprimed CXCR5+ CD8+ T cells contributes to high alloantibody and AMR in CCR5 KO recipient mice, which can be rescued with ACT.

Invariant NKT Cells Promote the Development of Highly Cytotoxic Multipotent CXCR3+CCR4+CD8+ T Cells That Mediate Rapid Hepatocyte Allograft Rejection.

Hepatocyte transplant represents a treatment for metabolic disorders but is limited by immunogenicity. Our prior work identified the critical role of CD8+ T cells, with or without CD4+ T cell help, in mediating hepatocyte rejection. In this study, we evaluated the influence of invariant NKT (iNKT) cells, uniquely abundant in the liver, upon CD8-mediated immune responses in the presence and absence of CD4+ T cells. To investigate this, C57BL/6 (wild-type) and iNKT-deficient Jα18 knockout mice (cohorts CD4 depleted) were transplanted with allogeneic hepatocytes. Recipients were evaluated for alloprimed CD8+ T cell subset composition, allocytotoxicity, and hepatocyte rejection. We found that CD8-mediated allocytotoxicity was significantly decreased in iNKT-deficient recipients and was restored by adoptive transfer of iNKT cells. In the absence of both iNKT cells and CD4+ T cells, CD8-mediated allocytotoxicity and hepatocyte rejection was abrogated. iNKT cells enhance the proportion of a novel subset of multipotent, alloprimed CXCR3+CCR4+CD8+ cytolytic T cells that develop after hepatocyte transplant and are abundant in the liver. Alloprimed CXCR3+CCR4+CD8+ T cells express cytotoxic effector molecules (perforin/granzyme and Fas ligand) and are distinguished from alloprimed CXCR3+CCR4-CD8+ T cells by a higher proportion of cells expressing TNF-α and IFN-γ. Furthermore, alloprimed CXCR3+CCR4+CD8+ T cells mediate higher allocytotoxicity and more rapid allograft rejection. Our data demonstrate the important role of iNKT cells in promoting the development of highly cytotoxic, multipotent CXCR3+CCR4+CD8+ T cells that mediate rapid rejection of allogeneic hepatocytes engrafted in the liver. Targeting iNKT cells may be an efficacious therapy to prevent rejection of intrahepatic cellular transplants.

Biochemical characterization of Borrelia burgdorferi's RecA protein.

RecA plays key roles in DNA recombination, replication and repair. Mutation of recA in the Lyme disease spirochete, Borrelia burgdorferi, fails to produce some of the phenotypes expected from study of recA mutation in other organisms. 'Missing' recA phenotypes include a lack of growth or viability effects, including in the presence of DNA damage, and a lack of a role in vlsE antigenic variation and infectivity. We present a purification and biochemical characterization of recombinant B. burgdorferi RecA protein. We find that B. burgdorferi RecA displays the expected properties of being a DNA-dependent ATPase, of having an intrinsic binding preference for ssDNA over dsDNA enhanced by ATP binding, of promoting DNA pairing and strand exchange reactions and of having a detectable coprotease activity with E. coli LexA repressor. DNA pairing and strand exchange reactions promoted by B. burgdorferi RecA show an unusually strong dependence upon the presence of the cognate ssDNA binding protein (SSB) but are very sensitive to inhibition by SSB when the ssDNA was prebound by SSB. This indicates B. burgdorferi RecA may have an enhanced requirement for recombinational mediators to promote RecA-SSB exchange, despite the absence of homologues of the RecF pathway proteins that normally play this role in eubacteria. Finally, we do not find any unusual, intrinsic properties of B. burgdorferi's RecA protein to explain the unusual phenotype of recA mutation and suggest that there may be alternative recombinase functions that could explain the 'missing' phenotypes.

The Borrelia burgdorferi telomere resolvase, ResT, possesses ATP-dependent DNA unwinding activity.

Spirochetes of the genus Borrelia possess unusual genomes harboring multiple linear and circular replicons. The linear replicons are terminated by covalently closed hairpin (hp) telomeres. Hairpin telomeres are formed from replicated intermediates by the telomere resolvase, ResT, in a phosphoryl transfer reaction with mechanistic similarities to those promoted by type 1B topoisomerases and tyrosine recombinases. There is growing evidence that ResT is multifunctional. Upon ResT depletion DNA replication unexpectedly ceases. Additionally, ResT possesses RecO-like biochemical activities being able to promote single-strand annealing on both free ssDNA and ssDNA complexed with cognate single-stranded DNA binding protein. We report here that ResT possesses DNA-dependent ATPase activity that promotes DNA unwinding with a 3΄-5΄ polarity. ResT can unwind a variety of substrates including synthetic replication forks and D-loops. We demonstrate that ResT's twin activities of DNA unwinding and annealing can drive regression of a model replication fork. These properties are similar to those of the RecQ helicase of the RecF pathway involved in DNA gap repair. We propose that ResT's combination of activities implicates it in replication and recombination processes operating on the linear chromosome and plasmids of Borrelia burgdorferi.

Novel humanized recombinant T cell receptor ligands protect the female brain after experimental stroke.

Transmigration of peripheral leukocytes to the brain is a major contributor to cerebral ischemic cell death mechanisms. Humanized partial major histocompatibility complex class II constructs (pMHC), covalently linked to myelin peptides, are effective for treating experimental stroke in males, but new evidence suggests that some inflammatory cell death mechanisms after brain injury are sex-specific. We here demonstrate that treatment with pMHC constructs also improves outcomes in female mice with middle cerebral artery occlusion (MCAO). HLA-DR2 transgenic female mice with MCAO were treated with RTL1000 (HLA-DR2 moiety linked to human MOG-35-55 peptide), HLA-DRa1-MOG-35-55, or vehicle (VEH) at 3, 24, 48, and 72 h after reperfusion and were recovered for 96 h or 2 weeks post-injury for measurement of histology (TTC staining) or behavioral testing. RTL1000- and DRa1-MOG-treated mice had profoundly reduced infarct volumes as compared to the VEH group, although higher doses of DRa1-MOG were needed for females vs. males evaluated previously. RTL1000-treated females also exhibited strongly improved functional recovery in a standard cylinder test. In novel studies of post-ischemic ultrasonic vocalization (USV), as measured by animal calls to their cage mates, we modeled in mice the post-stroke speech deficits common in human stroke survivors. The number of calls was reduced in injured animals relative to pre-MCAO baseline regardless of RTL1000 treatment status. However, call duration was significantly improved by RTL1000 treatment, suggesting benefit to the animal's recovery of vocalization capability. We conclude that both the parent RTL1000 molecule and the novel non-polymorphic DRα1-MOG-35-55 construct were highly effective immunotherapies for treatment of transient cerebral ischemia in females.