Francisco Victorino

Expression and Regulation of Chemokines in Murine and Human Type 1 Diabetes

More than one-half of the ~50 human chemokines have been associated with or implicated in the pathogenesis of type 1 diabetes, yet their actual expression patterns in the islet environment of type 1 diabetic patients remain, at present, poorly defined. Here, we have integrated a human islet culture system, murine models of virus-induced and spontaneous type 1 diabetes, and the histopathological examination of pancreata from diabetic organ donors with the goal of providing a foundation for the informed selection of potential therapeutic targets within the chemokine/receptor family. Chemokine (C-C motif) ligand (CCL) 5 (CCL5), CCL8, CCL22, chemokine (C-X-C motif) ligand (CXCL) 9 (CXCL9), CXCL10, and chemokine (C-X3-C motif) ligand (CX3CL) 1 (CX3CL1) were the major chemokines transcribed (in an inducible nitric oxide synthase–dependent but not nuclear factor-κB–dependent fashion) and translated by human islet cells in response to in vitro inflammatory stimuli. CXCL10 was identified as the dominant chemokine expressed in vivo in the islet environment of prediabetic animals and type 1 diabetic patients, whereas CCL5, CCL8, CXCL9, and CX3CL1 proteins were present at lower levels in the islets of both species. Of importance, additional expression of the same chemokines in human acinar tissues emphasizes an underappreciated involvement of the exocrine pancreas in the natural course of type 1 diabetes that will require consideration for additional type 1 diabetes pathogenesis and immune intervention studies.

Tissue-Resident NK Cells Mediate Ischemic Kidney Injury and Are Not Depleted by Anti-Asialo-GM1 Antibody.

NK cells are innate lymphoid cells important for immune surveillance, identifying and responding to stress, infection, and/or transformation. Whereas conventional NK (cNK) cells circulate systemically, many NK cells reside in tissues where they appear to be poised to locally regulate tissue function. In the present study, we tested the contribution of tissue-resident NK (trNK) cells to tissue homeostasis by studying ischemic injury in the mouse kidney. Parabiosis experiments demonstrate that the kidney contains a significant fraction of trNK cells under homeostatic conditions. Kidney trNK cells developed independent of NFIL3 and T-bet, and they expressed a distinct cell surface phenotype as compared with cNK cells. Among these, trNK cells had reduced asialo-GM1 (AsGM1) expression relative to cNK cells, a phenotype observed in trNK cells across multiple organs and mouse strains. Strikingly, anti–AsGM1 Ab treatment, commonly used as an NK cell–depleting regimen, resulted in a robust and selective depletion of cNKs, leaving trNKs largely intact. Using this differential depletion, we tested the relative contribution of cNK and trNK cells in ischemic kidney injury. Whereas anti–NK1.1 Ab effectively depleted both trNK and cNK cells and protected against ischemic/reperfusion injury, anti–AsGM1 Ab preferentially depleted cNK cells and failed to protect against injury. These data demonstrate unanticipated specificity of anti–AsGM1 Ab depletion on NK cell subsets and reveal a new approach to study the contributions of cNK and trNK cells in vivo. In total, these data demonstrate that trNK cells play a key role in modulating local responses to ischemic tissue injury in the kidney and potentially other organs.

Comprehensive assessment of chemokine expression profiles by flow cytometry

The chemokines are a large family of mainly secreted molecules involved in the regulation of numerous physiological and pathophysiological processes. Despite many years of investigation, the precise cellular sources of most chemokines have remained incompletely defined as a consequence of the limited availability of suitable reagents to visualize the expression of chemokine proteins at the single-cell level. Here, we developed a simple flow cytometry-based assay using commercially available chemokine-specific antibodies for efficient cell-associated detection of 37 of 39 murine chemokines. To demonstrate the utility of this methodology, we used it to reevaluate the nature of homeostatic chemokines in the hematopoietic compartment, to delineate the complete chemokine profiles of NK cells and B cells in response to major polyclonal stimuli, and to assess the chemokine response of DCs to bacterial infection. The versatility of this analytical methodology was further demonstrated by its application to selected human chemokines and should greatly facilitate any future investigation into chemokine biology at large.

Acute cardiac allograft rejection by directly cytotoxic CD4 T cells: parallel requirements for Fas and perforin.

Background. CD4 T cells can suffice as effector cells to mediate primary acute cardiac allograft rejection. Although CD4 T cells can readily kill appropriate target cells in vitro, the corresponding role of such cytolytic activity for mediating allograft rejection in vivo is unknown. Therefore, we determined whether the cytolytic effector molecules perforin (PFP) and/or FasL (CD95L) were necessary for CD4 T cell-mediated rejection in vivo. Methods. Wild-type C3H(H-2k) or Fas (CD95)-deficient C3Hlpr (H-2k) hearts were transplanted into immune-deficient C57B6rag−/− (H-2b) mice. Then, recipients were reconstituted with naïve purified CD4 T cells from wild-type, PFP-deficient, or FasL (gld)-deficient T-cell donors. Results. In vitro, alloreactive CD4 T cells were competent to lyse donor major histocompatibility complex class II+ target cells, largely by a Fas-dependent mechanism. In vivo, the individual disruption of donor Fas expression (lpr) or CD4 T-cell-derived PFP had no significant impact on acute rejection. However, FasL-deficient (gld) CD4 T cells demonstrated delayed allograft rejection. Importantly, the simultaneous removal of both donor Fas expression and CD4 T-cell PFP completely abrogated acute rejection, despite the persistence of CD4 T cells within the graft. Conclusions. Results demonstrate that the direct rejection of cardiac allografts by CD4 effector T cells requires the alternative contribution of graft Fas expression and T cell PFP expression. To our knowledge, this is the first demonstration that cytolytic activity by CD4 T cells can play an obligate role for primary acute allograft rejection in vivo.

Multiple layers of CD80/86-dependent costimulatory activity regulate primary, memory, and secondary lymphocytic choriomeningitis virus-specific T cell immunity.

ABSTRACT The lymphocytic choriomeningitis virus (LCMV) system constitutes one of the most widely used models for the study of infectious disease and the regulation of virus-specific T cell immunity. However, with respect to the activity of costimulatory and associated regulatory pathways, LCMV-specific T cell responses have long been regarded as relatively independent and thus distinct from the regulation of T cell immunity directed against many other viral pathogens. Here, we have reevaluated the contribution of CD28-CD80/86 costimulation in the LCMV system by use of CD80/86-deficient mice, and our results demonstrate that a disruption of CD28-CD80/86 signaling compromises the magnitude, phenotype, and/or functionality of LCMV-specific CD8+ and/or CD4+ T cell populations in all stages of the T cell response. Notably, a profound inhibition of secondary T cell immunity in LCMV-immune CD80/86-deficient mice emerged as a composite of both defective memory T cell development and a specific requirement for CD80 but not CD86 in the recall response, while a related experimental scenario of CD28-dependent yet CD80/86-independent secondary CD8+ T cell immunity suggests the existence of a CD28 ligand other than CD80/86. Furthermore, we provide evidence that regulatory T cells (TREGs), the homeostasis of which is altered in CD80/86−/− mice, contribute to restrained LCMV-specific CD8+ T cell responses in the presence of CD80/86. Our observations can therefore provide a more coherent perspective on CD28-CD80/86 costimulation in antiviral T cell immunity that positions the LCMV system within a shared context of multiple defects that virus-specific T cells acquire in the absence of CD28-CD80/86 costimulation.

High Efficiency of Antiviral CD4+ Killer T Cells

The destruction of infected cells by cytotxic T lymphocytes (CTL) is integral to the effective control of viral and bacterial diseases, and CTL function at large has long been regarded as a distinctive property of the CD8+T cell subset. In contrast, and despite their first description more than three decades ago, the precise contribution of cytotoxic CD4+T cells to the resolution of infectious diseases has remained a matter of debate. In particular, the CTL activity of pathogen-specific CD4+ “helper” T cells constitutes a single trait among a diverse array of other T cell functionalities, and overall appears considerably weaker than the cytolytic capacity of CD8+ effector T cells. Here, using an in vivo CTL assay, we report that cytotoxic CD4+T cells are readily generated against both viral and bacterial pathogens, and that the efficiency of MHC-II-restricted CD4+T cell killing adjusted for effector:target cell ratios, precise specificities and functional avidities is comparable in magnitude to that of CD8+T cells. In fact, the only difference between specific CD4+ and CD8+T cells pertains to the slightly delayed killing kinetics of the former demonstrating that potent CTL function is a cardinal property of both antiviral CD8+ and CD4+T cells.

Lysophosphatidic Acid Receptor 5 Inhibits B Cell Antigen Receptor Signaling and Antibody Response

Lysophospholipids have emerged as biologically important chemoattractants capable of directing lymphocyte development, trafficking, and localization. Lysophosphatidic acid (LPA) is a major lysophospholipid found systemically, and its levels are elevated in certain pathological settings, such as cancer and infections. In this study, we demonstrate that BCR signal transduction by mature murine B cells is inhibited upon LPA engagement of the LPA5 (GPR92) receptor via a Gα12/13-Arhgef1 pathway. The inhibition of BCR signaling by LPA5 manifests by impaired intracellular calcium store release and most likely by interfering with inositol 1,4,5-triphosphate receptor activity. We further show that LPA5 also limits Ag-specific induction of CD69 and CD86 expression and that LPA5-deficient B cells display enhanced Ab responses. Thus, these data show that LPA5 negatively regulates BCR signaling, B cell activation, and immune response. Our findings extend the influence of lysophospholipids on immune function and suggest that alterations in LPA levels likely influence adaptive humoral immunity.

Prolongation of cardiac allograft survival by a novel population of autologous CD117+ bone marrow-derived progenitor cells

Autologous CD117+ progenitor cells (PC) have been successfully utilized in myocardial infarction and ischemic injury, potentially through the replacement/repair of damaged vascular endothelium. To date, such cells have not been used to enhance solid organ transplant outcome. In this study, we determined whether autologous bone marrow‐derived CD117+PC could benefit cardiac allograft survival, possibly by replacing donor vascular cells. Autologous, positively selected CD117+PC were administered posttransplantation and allografts were assessed for acute rejection. Although significant generation of recipient vascular cell chimerism was not observed, transferred PC disseminated both to the allograft and to peripheral lymphoid tissues and facilitated a significant, dose‐dependent prolongation of allograft survival. While CD117+PC dramatically inhibited alloreactive T cell proliferation in vitro, this property did not differ from nonprotective CD117− bone marrow populations. In vivo, CD117+ PC did not significantly inhibit T cell alloreactivity or increase peripheral regulatory T cell numbers. Thus, rather than inhibiting adaptive immunity to the allograft, CD117+ PC may play a cytoprotective role in prolonging graft survival. Importantly, autologous CD117+PC appear to be distinct from bone marrow‐derived mesenchymal stem cells (MSC) previously used to prolong allograft survival. As such, autologous CD117+PC represent a novel cellular therapy for promoting allograft survival.

Aging Boosts Antiviral CD8+T Cell Memory Through Improved Engagement Of Diversified Recall Response Determinants

The determinants of protective CD8+ memory T cell (CD8+TM) immunity remain incompletely defined and may in fact constitute an evolving agency as aging CD8+TM progressively acquire enhanced rather than impaired recall capacities. Here, we show that old as compared to young antiviral CD8+TM more effectively harness disparate molecular processes (cytokine signaling, trafficking, effector functions, and co-stimulation/inhibition) that in concert confer greater secondary reactivity. The relative reliance on these pathways is contingent on the nature of the secondary challenge (greater for chronic than acute viral infections) and over time, aging CD8+TM re-establish a dependence on the same accessory signals required for effective priming of naïve CD8+T cells in the first place. Thus, our findings are consistent with the recently proposed “rebound model” that stipulates a gradual alignment of naïve and CD8+TM properties, and identify a diversified collection of potential targets that may be exploited for the therapeutic modulation of CD8+TM immunity.