Andrew Zloza

Oncolytic viruses: a new class of immunotherapy drugs

Oncolytic viruses represent a new class of therapeutic agents that promote anti-tumour responses through a dual mechanism of action that is dependent on selective tumour cell killing and the induction of systemic anti-tumour immunity. The molecular and cellular mechanisms of action are not fully elucidated but are likely to depend on viral replication within transformed cells, induction of primary cell death, interaction with tumour cell antiviral elements and initiation of innate and adaptive anti-tumour immunity. A variety of native and genetically modified viruses have been developed as oncolytic agents, and the approval of the first oncolytic virus by the US Food and Drug Administration (FDA) is anticipated in the near future. This Review provides a comprehensive overview of the basic biology supporting oncolytic viruses as cancer therapeutic agents, describes oncolytic viruses in advanced clinical trials and discusses the unique challenges in the development of oncolytic viruses as a new class of drugs for the treatment of cancer.

Pro-inflammatory chemokine CCL2 (MCP-1) promotes healing in diabetic wounds by restoring the macrophage response.

Prior studies suggest that the impaired healing seen in diabetic wounds derives from a state of persistent hyper-inflammation characterized by harmful increases in inflammatory leukocytes including macrophages. However, such studies have focused on wounds at later time points (day 10 or older), and very little attention has been given to the dynamics of macrophage responses in diabetic wounds early after injury. Given the importance of macrophages for the process of healing, we studied the dynamics of macrophage response during early and late phases of healing in diabetic wounds. Here, we report that early after injury, the diabetic wound exhibits a significant delay in macrophage infiltration. The delay in the macrophage response in diabetic wounds results from reduced Chemokine (C-C motif) ligand 2 (CCL2) expression. Importantly, one-time treatment with chemoattractant CCL2 significantly stimulated healing in diabetic wounds by restoring the macrophage response. Our data demonstrate that, rather than a hyper-inflammatory state; the early diabetic wound exhibits a paradoxical and damaging decrease in essential macrophage response. Our studies suggest that the restoration of the proper kinetics of macrophage response may be able to jumpstart subsequent healing stages. CCL2 chemokine-based therapy may be an attractive strategy to promote healing in diabetic wounds.

Active β-Catenin Signaling Is an Inhibitory Pathway for Human Immunodeficiency Virus Replication in Peripheral Blood Mononuclear Cells

ABSTRACT The Wnt/β-catenin pathway is involved in cell functions governing development and disease. In modeling postentry restriction of human immunodeficiency virus (HIV) replication in astrocytes, we reported that part of this natural resistance to productive replication of HIV in astrocytes involved expression of proteins of the Wnt/β-catenin signaling pathway. We determined here whether induction of β-catenin signaling in peripheral blood mononuclear cells (PBMCs) can modulate HIV replication. Given that lithium is an inducer of β-catenin signaling, we used it as a tool to determine the impact of β-catenin signaling on HIV replication in PBMCs. We demonstrated that lithium inhibited the replication of T-tropic and primary isolates of HIV by >90% and did so in noncytotoxic/noncytostatic concentrations and in a β-catenin-dependent manner. Specifically, inhibiting β-catenin signaling by transfection of dominant-negative mutant constructs to either T-cell factor 4, the downstream effector of Wnt signaling, or β-catenin, the central mediator of this pathway, abrogated the ability of lithium to inhibit HIV replication. Moreover, when Wnt/β-catenin signaling was inhibited, the level of HIV replication was enhanced by fourfold. To confirm the in vivo relevance of the β-catenin pathway in repressing HIV replication, we evaluated HIV-positive antiretroviral therapy-naive patients who were on lithium therapy. These patients demonstrated a reduction in viral load, which increased as the dose of lithium was reduced. Collectively, these data indicate that β-catenin signaling is an intrinsic molecular pathway restricting HIV replication in PBMCs.

Antigenic peptide nanofibers elicit adjuvant-free CD8 + T cell responses

Vaccines that elicit robust CD8⁺ T cell responses are desirable for protection against infectious diseases and cancers. However, most vaccine adjuvants fail to elicit robust CD8⁺ T cell responses without inflammation and associated toxicity. We recently reported that self-assembling peptides that form nanofibers in physiological buffers elicited strong adjuvant-free and antigen-specific antibody responses in mice. However, whether or not such nanofibers likewise can elicit strong CD8⁺ T cell responses is unknown. Here, we demonstrate that the self-assembling peptide Q11 conjugated to a CD8⁺ T cell epitope of ovalbumin (Q11-OVA), elicits strong antigen-specific primary and recall responses, and in a vaccination regimen protects against subsequent infection. Importantly, we show that these antigenic peptide nanofibers do not persist as an inflammatory antigen depot at the injection site. Our results demonstrate for the first time that self-assembling peptides may be useful as carriers for vaccines where CD8⁺ T cell-mediated protection is needed.

Mutant HSP70 Reverses Autoimmune Depigmentation in Vitiligo

Vitiligo can be reversed through immune targeting with mutant heat shock protein 70. New Treatment Makes Vitiligo Beat It Whether your grant application is due, you have a paper that needs to be submitted, or your patient load is too high, medical science is not a relaxing profession. High stress is known to negatively affect your health at both the whole body and cellular level. One way the body responds to cellular stressors is through the induction of heat shock proteins (HSPs). Now, Mosenson et al. suggest that mutant HSP70 could be a potential treatment for autoimmune vitiligo. The authors noticed that mutant inducible HSP70 (HSP70i) could prevent T cell–mediated depigmentation in a mouse model of vitiligo, perhaps by shifting dendritic cells from an inflammatory to a regulatory phenotype. Moreover, a DNA vaccine of the mutant HSP70i could be used therapeutically to partially restore pigmentation in a second model of depigmentation. The authors then took these studies into ex vivo human skin, showing that their mutant HSP70i could prevent the disease-related shift from quiescent to effector T cell phenotype. Although these observations still need to be translated into the clinic, they form the basis for a new potential treatment for autoimmune vitiligo. Vitiligo is an autoimmune disease characterized by destruction of melanocytes, leaving 0.5% of the population with progressive depigmentation. Current treatments offer limited efficacy. We report that modified inducible heat shock protein 70 (HSP70i) prevents T cell–mediated depigmentation. HSP70i is the molecular link between stress and the resultant immune response. We previously showed that HSP70i induces an inflammatory dendritic cell (DC) phenotype and is necessary for depigmentation in vitiligo mouse models. Here, we observed a similar DC inflammatory phenotype in vitiligo patients. In a mouse model of depigmentation, DNA vaccination with a melanocyte antigen and the carboxyl terminus of HSP70i was sufficient to drive autoimmunity. Mutational analysis of the HSP70i substrate-binding domain established the peptide QPGVLIQVYEG as invaluable for DC activation, and mutant HSP70i could not induce depigmentation. Moreover, mutant HSP70iQ435A bound human DCs and reduced their activation, as well as induced a shift from inflammatory to tolerogenic DCs in mice. HSP70iQ435A-encoding DNA applied months before spontaneous depigmentation prevented vitiligo in mice expressing a transgenic, melanocyte-reactive T cell receptor. Furthermore, use of HSP70iQ435A therapeutically in a different, rapidly depigmenting model after loss of differentiated melanocytes resulted in 76% recovery of pigmentation. Treatment also prevented relevant T cells from populating mouse skin. In addition, ex vivo treatment of human skin averted the disease-related shift from quiescent to effector T cell phenotype. Thus, HSP70iQ435A DNA delivery may offer potent treatment opportunities for vitiligo.

Bone marrow-derived immature myeloid cells are a main source of circulating suPAR contributing to proteinuric kidney disease

Excess levels of protein in urine (proteinuria) is a hallmark of kidney disease that typically occurs in conjunction with diabetes, hypertension, gene mutations, toxins or infections but may also be of unknown cause (idiopathic). Systemic soluble urokinase plasminogen activator receptor (suPAR) is a circulating factor implicated in the onset and progression of chronic kidney disease (CKD), such as focal segmental glomerulosclerosis (FSGS). The cellular source(s) of elevated suPAR associated with future and progressing kidney disease is unclear, but is likely extra-renal, as the pathological uPAR is circulating and FSGS can recur even after a damaged kidney is replaced with a healthy donor organ. Here we report that bone marrow (BM) Gr-1lo immature myeloid cells are responsible for the elevated, pathological levels of suPAR, as evidenced by BM chimera and BM ablation and cell transfer studies. A marked increase of Gr-1lo myeloid cells was commonly found in the BM of proteinuric animals having high suPAR, and these cells efficiently transmit proteinuria when transferred to healthy mice. In accordance with the results seen in suPAR-associated proteinuric animal models, in which kidney damage is caused not by local podocyte-selective injury but more likely by systemic insults, a humanized xenograft model of FSGS resulted in an expansion of Gr-1lo cells in the BM, leading to high plasma suPAR and proteinuric kidney disease. Together, these results identify suPAR as a functional connection between the BM and the kidney, and they implicate BM immature myeloid cells as a key contributor to glomerular dysfunction.

NKG2D signaling on CD8 + T cells represses T-bet and rescues CD4-unhelped CD8 + T cell memory recall but not effector responses

CD4-unhelped CD8+ T cells are functionally defective T cells primed in the absence of CD4+ T cell help. Given the co-stimulatory role of natural-killer group 2, member D protein (NKG2D) on CD8+ T cells, we investigated its ability to rescue these immunologically impotent cells. We demonstrate that augmented co-stimulation through NKG2D during priming paradoxically rescues memory, but not effector, CD8+ T cell responses. NKG2D-mediated rescue is characterized by reversal of elevated transcription factor T-box expressed in T cells (T-bet) expression and recovery of interleukin-2 and interferon-γ production and cytolytic responses. Rescue is abrogated in CD8+ T cells lacking NKG2D. Augmented co-stimulation through NKG2D confers a high rate of survival to mice lacking CD4+ T cells in a CD4-dependent influenza model and rescues HIV-specific CD8+ T cell responses from CD4-deficient HIV-positive donors. These findings demonstrate that augmented co-stimulation through NKG2D is effective in rescuing CD4-unhelped CD8+ T cells from their pathophysiological fate and may provide therapeutic benefits.

HSP70i is a critical component of the immune response leading to vitiligo

HSP70i and other stress proteins have been used in anti‐tumor vaccines. This begs the question whether HSP70i plays a unique role in immune activation. We vaccinated inducible HSP70i (Hsp70‐1) knockout mice and wild‐type animals with optimized TRP‐1, a highly immunogenic melanosomal target molecule. We were unable to induce robust and lasting depigmentation in the Hsp70‐1 knockout mice, and in vivo cytolytic assays revealed a lack of cytotoxic T‐lymphocyte activity. Absence of T‐cell infiltration to the skin and maintenance of hair follicle melanocytes were observed. By contrast, depigmentation proceeded without interruption in mice lacking a tissue‐specific constitutive isoform of HSP70 (Hsp70‐2) vaccinated with TRP‐2. Next, we demonstrated that HSP70i was necessary and sufficient to accelerate depigmentation in vitiligo‐prone Pmel‐1 mice, accompanied by lasting phenotypic changes in dendritic cell subpopulations. In summary, these studies assign a unique function to HSP70i in vitiligo and identify HSP70i as a targetable entity for treatment.

Potent HIV-specific responses are enriched in a unique subset of CD8+ T cells that coexpresses CD4 on its surface

In humans, approximately 3% of peripheral CD8+ T cells coexpress CD4 dimly on their surface and hence are designated as CD4(dim)CD8(bright) T cells. We evaluated the contribution of this CD4(dim)CD8(bright) T-cell population to anti-HIV immunity. We demonstrate that CD4(dim)CD8(bright) T cells generate greater than 55% of CD8+ T-cell antigen recognition and effector response to HIV, as evaluated by multiple parameters for assessing T-cell antiviral immunity, including HIV tetramer recognition, cytokine production, and cytolytic potential. Inhibition of major histocompatibility class II (MHC-II) on target cells or CD4 on CD4(dim)CD8(bright) T cells diminishes their anti-HIV responses, suggesting that CD4 on effector cells and MHC-II on target cells provides an additional arm of contact between effector and target cells which is critical to CD4(dim)CD8(bright) T-cell function. CD4(dim)CD8(bright) T cells also exhibit features that are indicative of central memory T cells. Finally, CD4(dim)CD8(bright) T cells are elevated in blood of HIV+ long-term nonprogressors in comparison to HIV- donors. Collectively, our findings show that CD4(dim)CD8(bright) T cells designate an enriched antiviral subpopulation of CD8+ T cells that should be targeted for therapeutic intervention or evaluation of vaccine efficacy.

Multiple populations of T lymphocytes are distinguished by the level of CD4 and CD8 coexpression and require individual consideration.

Expression of CD4 or CD8 on T cells is pivotal in defining them as T helper or T cytotoxic cells, respectively. Aside from an intermediate stage during T cell maturation in the thymus, CD4 and CD8 molecules were thought to be mutually exclusive on mature T cells. However, accumulating evidence has proven that these two molecules can be coexpressed on mature T cells [1–7]. Populations of CD8 T cells re-expressing CD4 have been designated CD4CD8 or CD4CD8 T cells, and likewise, populations of CD4 T cells re-expressing CD8 have been designated CD4CD8 or CD4CD8 T cells. Much of what we know about CD4 CD8 T cells is based on literature that has concentrated on the CD4CD8 T cell population. These studies collectively established the importance of studying CD4 CD8 T cell populations through demonstrating that the CD4 molecule expressed on CD8 T cells is functional, as indicated by several lines of evidence, including the ability to coprecipitate CD4 with the Scr family protein tyrosine kinase p56lck [1] and the ability of CD4 on CD8 T cells to mediate chemotaxis in response to interleukin-16, a CD4-specific ligand [3]. We have shown that CD4CD8 T cells recognize viral [cytomegalovirus (CMV)] antigen more potently than CD8 T cells, which do not re-express CD4 on their surface (CD4CD8 T cells) [6]. Recent data suggest that CD4CD8 T cells function as CD8 effector cells, as assessed in the severe combined immunodeficient (SCID) model of lymphocytic choriomeningitis virus (LCMV) and allogenic immunity [8, 9] and in the chimpanzee model of hepatitis C virus immunity [7]. In analyzing the role of CD4 on CD8 T cells and the biologic significance of this cell population, it is critical that these cells are defined appropriately and that no contaminating populations are included in the analysis. Based on a limited number of markers shown to be shared by both populations, some investigators have combined the analysis of CD4CD8 and CD4CD8 T cells as one population, defining all double-positive (CD4 CD8 ) T cells [7]. Combining these populations to deduce functional potential is problematic. Our own analyses, when collecting a minimum of 100,000 events per sample for immunostaining and using CD4 antibodies conjugated to phycoerythrin (PE) or allophycocyanin (APC), demonstrate that multiple subpopulations of T cells, defined by the level of their expression of CD4 and CD8, exist within the CD4 CD8 double-positive T cell population. As seen in Figure 1A, based on the intensity of CD4 and CD8 expression, six different populations are apparent: the singlepositive populations include CD8 CD4 (denoted as P1) and CD4 CD8 (denoted as P6), and the double-positive populations include CD4CD8 (denoted as P2), CD4CD8 (denoted as P3), CD4 CD8 (denoted as P4), and CD4CD8 (denoted as P5). Given that NKT cells can be CD4 CD8 , CD4CD8, CD4CD8 , or CD4 CD8, it is possible that CD4 CD8 NKT cells may be present within the subpopulations of CD4 CD8 T cells to varying degrees. Therefore, we examined the frequency of NKT cells within the CD4 CD8 T cell subpopulations. We demonstrate that invariant NKT cells, defined by coexpression of CD3 and 6B11 [10], and non-invariant NKT cells, defined by coexpression of CD3 and CD16/56, are present within these CD4 CD8 T cell subpopulations at varying frequencies (Fig. 1, C and D). Expression of invariant NKT and non-invariant NKT markers is most evident in the CD4CD8 (P5) subpopulation, where up to 29% of the cells were found to be invariant CD3 6B11 NKT cells, and up to 26% of the cells were found to be non-invariant CD3 CD16/56 NKT cells. Our studies here show that in characterizing the role of cells coexpressing CD4 and CD8, it is critical that multiple CD4 CD8 T cell subpopulations (CD4CD8, CD4CD8, CD4 CD8 , and CD4CD8) are not evaluated together as one double-positive population. Combining these populations, which may not share similar characteristics and may express differentiation and activation surface markers to varying degrees, and not depleting certain populations of contaminating cells (i.e., NKT cells) will mask the lineage and functional potential analysis of each individual subpopulation of CD4 CD8 T cells.