CD49d provides access to "untouched" human Foxp3+ Treg free of contaminating effector cells

The adoptive transfer of regulatory Foxp3(+) T (Treg) cells has been shown in various animal models to prevent inflammatory immune and autoimmune diseases. Translation into therapeutic applications, however, is hindered by the lack of suitable techniques and markers. CD25, commonly used to isolate Treg cells from mice, has only limited value in humans as it is also present on proinflammatory CD4(+) effector cells. Here we show that clean populations of human Foxp3(+) Treg cells can be obtained with antibodies directed against CD49d. The marker is present on proinflammatory peripheral blood mononuclear cells but is absent on immune-suppressive Treg cells. Depletion with alpha-CD49d removes contaminating interferon-gamma (IFN-gamma)- and interleukin-17 (IL-17)-secreting cells from Treg preparations of CD4(+)CD25(high) cells. More importantly, in combination with alpha-CD127 it allows the isolation of "untouched" Foxp3(+) Treg (ie, cells that have not been targeted by an antibody during purification). The removal of CD49d(+)/CD127(+) cells leaves a population of Foxp3(+) Treg virtually free of contaminating CD25(+) effector cells. The cells can be expanded in vitro and are effective suppressors both in vitro and in vivo. Thus, CD49d provides access to highly pure populations of untouched Foxp3(+) Treg cells conferring maximal safety for future clinical applications.     

Borderline personality organization and psychopathic traits in nonclinical adolescents: relationships of identity diffusion, primitive defense mechanisms and reality testing with callousness and impulsivity traits

Although psychotherapeutic observation and empirical data suggest a link between borderline and antisocial personality disorder or traits in adolescents, there is no study on the relationships of borderline personality organization (BPO) and psychopathic traits in adolescents. The aim of this study was to explore the relationship of structural criteria of (BPO) as assessed by the French version of the Borderline Personality Inventory (BPI), with psychopathic traits, as assessed by the French version of the Levenson Self-Report Psychopathy Scale (LSRP), in a nonclinical sample of 243 adolescents. Significant correlations were found between the BPI scales of identity diffusion, primitive defense mechanisms, impaired reality testing, and psychopathic traits of callousness and impulsivity, suggesting that BPO may contribute to psychopathic traits in nonforensic, nonclinical adolescents.     

Patterns of interpersonal problems and their improvement in depressive and anxious patients treated with psychoanalytic therapy

Interpersonal problems were studied in 121 patients treated with psychoanalytic therapy using the Inventory of Interpersonal Problems. Four characteristic subtypes were identified, which differed in the quality and flexibility of their interpersonal behavior. Independent of the predominant type of interpersonal problems, the psychotherapy treatment led to strong decreases in interpersonal distress and increases in interpersonal differentiation. Psychoanalytic therapy was highly effective for all identified interpersonal subtypes and seems to help patients achieve more satisfactory relationships.     

Recurrence of Type 1 Diabetes After Simultaneous Pancreas-Kidney Transplantation, Despite Immunosuppression, Is Associated With Autoantibodies and Pathogenic Autoreactive CD4 T-Cells

OBJECTIVE

To investigate if recurrent autoimmunity explained hyperglycemia and C-peptide loss in three immunosuppressed simultaneous pancreas-kidney (SPK) transplant recipients.

RESEARCH DESIGN AND METHODS

We monitored autoantibodies and autoreactive T-cells (using tetramers) and performed biopsy. The function of autoreactive T-cells was studied with in vitro and in vivo assays.

RESULTS

Autoantibodies were present pretransplant and persisted on follow-up in one patient. They appeared years after transplantation but before the development of hyperglycemia in the remaining patients. Pancreas transplant biopsies were taken within ∼1 year from hyperglycemia recurrence and revealed β-cell loss and insulitis. We studied autoreactive T-cells from the time of biopsy and repeatedly demonstrated their presence on further follow-up, together with autoantibodies. Treatment with T-cell–directed therapies (thymoglobulin and daclizumab, all patients), alone or with the addition of B-cell–directed therapy (rituximab, two patients), nonspecifically depleted T-cells and was associated with C-peptide secretion for >1 year. Autoreactive T-cells with the same autoantigen specificity and conserved T-cell receptor later reappeared with further C-peptide loss over the next 2 years. Purified autoreactive CD4 T-cells from two patients were cotransplanted with HLA-mismatched human islets into immunodeficient mice. Grafts showed β-cell loss in mice receiving autoreactive T-cells but not control T-cells.

CONCLUSIONS

We demonstrate the cardinal features of recurrent autoimmunity in three such patients, including the reappearance of CD4 T-cells capable of mediating β-cell destruction. Markers of autoimmunity can help diagnose this underappreciated cause of graft loss. Immune monitoring during therapy showed that autoimmunity was not resolved by the immunosuppressive agents used.Type 1 diabetes is an autoimmune disease characterized by the lymphocytic infiltration of the pancreatic islets (insulitis), β-cell destruction, and loss of insulin secretion (1). Autoreactive CD4 and CD8 T-cells and autoantibodies to islet cell autoantigens are detected in patients and pre-diabetic subjects, often preceding diabetes onset by months to years. Insulin, GAD (GAD, 65-kDa isoform), the tyrosine-like phosphatase protein IA-2, the islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), and the recently identified cation efflux transporter ZnT8 are well characterized and commonly targeted autoantigens (2–8).Simultaneous pancreas-kidney (SPK) transplantation from deceased donors restores insulin secretion in patients and corrects end-stage renal disease (9). Immunological failures occur in a minority of transplant recipients and are usually categorized as chronic rejection. Another possible cause of immunological failure is recurrence of type 1 diabetes. This was initially reported a few weeks after transplantation in recipients of the tail of the pancreas from living-related HLA-identical twins or siblings who, because of HLA matching, received either no or reduced immunosuppression (10–13). However, diabetes recurrence was <10% in a large series of recipients of deceased donor grafts given immunosuppression sufficient to prevent rejection (14). Further studies associated islet cell autoantibodies with graft failure (15–19) but lacked biopsy data, and rejection was not excluded. Two SPK recipients had partial evidence for diabetes recurrence (20), including limited biopsy data showing selective β-cell loss and/or insulitis and limited autoantibody data (20). None of these studies assessed autoantigen-specific T-cells in the context of graft loss. Islet autoimmunity is considered rare and is not routinely monitored in SPK recipients. Thus, recurrence of type 1 diabetes in SPK recipients remains incompletely characterized.We investigated whether recurrent islet autoimmunity explained the hyperglycemia and loss of insulin secretion observed in three immunosuppressed SPK recipients in the absence of rejection. The immunological assessment included both retrospective and prospective testing for autoantibodies and prospective testing for autoantigen-specific T-cells. Monitoring was continued on extended follow-up after patients were diagnosed with recurrence of type 1 diabetes and received additional immunotherapy to antagonize the autoimmune process. We also characterized the functional features of the autoreactive T-cells detected in these patients in the context of recurring diabetes, using both in vitro and in vivo experimental assays to test the pathogenic effects of the autoreactive T-cells.     

Long-term tripotent differentiation capacity of human neural stem (NS) cells in adherent culture

Stem cell lines that provide a renewable and scaleable supply of central nervous system cell types would constitute an invaluable resource for basic and applied neurobiology. Here we describe the generation and long-term expansion of multiple human foetal neural stem (NS) cell lines in monolayer culture without genetic immortalization. Adherent human NS cells are propagated in the presence of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), under which conditions they stably express neural precursor markers and exhibit negligible differentiation into neurons or glia. However, they produce astrocytes, oligodendrocytes, and neurons upon exposure to appropriate differentiation factors. Single cell cloning demonstrates that human NS cells are tripotent. They retain a diploid karyotype and constant neurogenic capacity after over 100 generations. In contrast to human neurospheres, we observe no requirement for the cytokine leukaemia inhibitory factor (LIF) for continued expansion of adherent human NS cells. Human NS cells can be stably transfected to provide reporter lines and readily imaged in live monolayer cultures, creating the potential for high content genetic and chemical screens.