CAR γδ T cells for cancer immunotherapy. Is the field more yellow than green?

Engineered immune cell therapy to treat malignancies refractory to conventional therapies is modernizing oncology. Although αß T cells are time-tested chassis for CAR, potential graft versus host disease (GvHD) apart from cytokine toxicity and antigen escape pose limitations to this approach. αß T cell malignancy challenges isolation and expansion of therapeutic T cells. Moreover, αß T cells may pose toxicity risk to inflammation sensitive vital tissues bearing the tumor. The HLA independent, multivalent, versatile and systemic anti-tumor immunity increases the desirability of γδ T cells as an alternate chassis for CAR. Indeed, CD19 γδ CAR T cell therapy to treat advanced lymphoma reached a milestone with the fast track status by FDA. However, reduced tumor-toxicity, homing, in vivo persistence and heterogeneity limits the translation of this therapy. The field is gaining momentum in recent years with optimization of gene delivery approaches and mechanistic insights into co-signaling requirements in γδ T cells. There is a renewed interest in customizing design of CAR guided by the biology of the host immune cells. Progress has been made in the current good manufacturing practice compatible expansion and engineering protocols for the δ1 and δ2 T cells. γδ CAR T cells may find its niche in the clinical situations wherein conventional CAR therapy is less suitable due to propensity for cytokine toxicity or off-tumor effect. As the therapy is moving towards clinical trials, this review chronicles the hitherto progress in the therapeutic engineering of γδ T cells for cancer immunotherapy.

Engineering off-the-shelf universal CAR T cells: A silver lining in the cloud.

CAR therapy holds promise in treating aggressive hematological malignancies. Nonetheless, the present autologous CAR therapy regimen makes multiple patients ineligible for the therapy due to inadequate quantity, quality and purity of the product. Furthermore, timely manufacturing of benchmarked cell products is logistically challenging and unaffordable. Extensive genetic modifications may be required to overcome the biological, clinical and manufacturing limitations of the autologous CAR therapy. n the light of the numerous configurations of CAR therapy, engineering "off-the-shelf" universal CAR T cells (UCART) is emerging as a safer, effective and affordable alternative to conventional CAR T cells With UCART therapy, batch production of a quality-controlled product with multiplex genetic modification can be feasible in a shorter period of time. Currently vast majority of the UCART programs target CD19 followed by BCMA and CD70. In order to make universal CAR T cell therapy possible, it is imperative to have engineering strategies to curb graft versus host disease (GvHD) and graft rejection (GR). Moreover, approaches to offer alternate strategies for intense preparative chemotherapy, infection control and CAR T cell persistence need to be optimized. An ideal universal immune receptor (UIR) design should counter the antigen escape and further the therapeutic value and affordability. UIRs would allow flexibility to personalize the therapy based on the specific malignancy characteristics as well. With the innovations in the inducible molecular switch, split CAR design, CRISPR/Cas9 mediated gene targeting, rational subset composition and cryopreservation, the strategies to engineer universal CAR T therapy is fast advancing from bench to bedside.

Cytokines in the generation and function of regulatory T cell subsets in leishmaniasis.

CD4+ T regulatory cells (Tregs) are a group of T lymphocytes that maintain self-tolerance and protect the host from inflammation-induced tissue damage. An interacting network of cytokines and transcription factors influence the origin, differentiation, and function of the Tregs in primary and secondary lymphoid organs. However, following antigenic stimulation, it can also be induced at the sites of infection. Immune cell resident microbial pathogens, such as Leishmania, employ varieties of mechanisms to promote the suppressive functions of Tregs for protective evasion from the host immune system. This establishes a state of immune unresponsiveness in the host, exacerbating the disease in Leishmania infection. Elimination of Leishmania pathogens is accomplished with a strong pro-inflammatory response accompanied by the release of host protective cytokines such as Interleukin-2 (IL-2), Interferon-gamma (IFN-γ), and Tumor necrosis factor-alpha (TNF-α), which functions through suppression of Tregs or making the effector cells recalcitrant to Treg mediated suppression. Nevertheless, during chronic infection, the persistence of unwarranted pro-inflammatory cytokines can trigger self-tissue damage. Tregs limit the consequence of chronic inflammation to restrict self-harm suggesting its mutually opposing role in host protection. Furthermore, Tregs function to prevent complete parasite clearance to provide long-term immunity to re-infection. This review summarizes the roles of pro-inflammatory and anti-inflammatory cytokines involved in the homing, activation, differentiation, and suppression of Tregs in the course of Leishmania infection. We also suggest cytokines that can be modulated as potential therapeutic targets to treat Leishmania infection.

Identification and characterization of an alternative cancer-derived PD-L1 splice variant.

Therapeutic blockade of the PD-1/PD-L1 axis is recognized as an effective treatment for numerous cancer types. However, only a subset of patients respond to this treatment, warranting a greater understanding of the biological mechanisms driving immune evasion via PD-1/PD-L1 signaling and other T-cell suppressive pathways. We previously identified a head and neck squamous cell carcinoma with human papillomavirus integration in the PD-L1 locus upstream of the transmembrane domain-encoding region, suggesting expression of a truncated form of PD-L1 (Parfenov et al., Proc Natl Acad Sci USA 111(43):15544-15549, 2014). In this study, we extended this observation by performing a computational analysis of 33 other cancer types as well as human cancer cell lines, and identified additional PD-L1 isoforms with an exon 4 enrichment expressed in 20 cancers and human cancer cell lines. We demonstrate that cancer cell lines with high expression levels of exon 4-enriched PD-L1 generate a secreted form of PD-L1. Further biochemical studies of exon 4-enriched PD-L1 demonstrated that this form is secreted and maintains the capacity to bind PD-1 as well as to serve as a negative regulator on T cell function, as measured by inhibition of IL-2 and IFNg secretion. Overall, we have demonstrated that truncated forms of PD-L1 exist in numerous cancer types, and have validated that truncated PD-L1 can be secreted and negatively regulate T cell function.

Cutting Edge: a novel, human-specific interacting protein couples FOXP3 to a chromatin-remodeling complex that contains KAP1/TRIM28.

Regulatory T cells (Tregs) play a pivotal role in the maintenance of immunological self-tolerance. Deficiency or dysfunction of Tregs leads to severe autoimmune diseases. Although the forkhead/winged-helix family member FOXP3 is critical for Treg differentiation and function, the molecular basis for FOXP3 function remains unclear. In this study, we identified and characterized a human-specific FOXP3-interacting protein, referred to as FIK (FOXP3-interacting KRAB domain-containing protein). FIK is highly expressed in Tregs and acts as a bridging molecule to link FOXP3 with the chromatin-remodeling scaffold protein KAP1 (TIF-1ß/TRIM28). Disruption of the FOXP3-FIK-KAP1 complex in Tregs restored expression of FOXP3-target genes and abrogated the suppressor activity of the Tregs. These data demonstrate a critical role for FIK in regulating FOXP3 activity and Treg function.

Gut microbiome homeostasis and the future of probiotics in cancer immunotherapy.

The gut microbiome has an impact on cancer immune surveillance and immunotherapy, with recent studies showing categorical differences between immunotherapy-sensitive and immunotherapy-resistant cancer patient cohorts. Although probiotics are traditionally being supplemented to promote treatments or sustain therapeutic benefits; the FDA has not approved any for use with immunotherapy. The first step in developing probiotics for immunotherapy is identifying helpful or harmful bacteria down to the strain level. The gut microbiome's heterogeneity before and during treatment is also being investigated to determine microbial strains that are important for immunotherapy. Moreover, Dietary fiber intake, prebiotic supplementation and fecal microbiota transplantation (FMT) were found to enhance intratumoral CD8+ T cell to T-reg ratio in the clinics. The possibility of probiotic immunotherapy as a "living adjuvant" to CAR treatment and checkpoint blockade resistance is actively being investigated.

CD40 expression levels modulate regulatory T cells in Leishmania donovani infection.

Dendritic cell (DC)-expressed CD40 is shown to play crucial roles in eliciting effector T cell responses, primarily the proinflammatory CD4(+) Th subsets and cytotoxic CD8(+) T cells that eliminate various infections and tumors, respectively. In contrast, DCs are also implied in the generation of regulatory T cells (Tregs) that counteract the functions of the proinflammatory Th subsets and exacerbate infections. However, the role of DC-expressed CD40 in the generation of Tregs is unknown. In this study, we generated bone marrow-derived DCs from mice (on a BALB/c background) expressing different levels of CD40 and tested their relative efficiency in generating Tregs. We observed that low levels of CD40 expression were required for efficient Treg generation. DCs expressing low levels of CD40 induced Tregs, whereas DCs expressing high levels of CD40 induced effector T cells, possibly CD8(+)CD40(+) T cells with a contraregulatory activity; the adoptive transfer of the former DC exacerbated whereas the latter significantly reduced Leishmania donovani infection in BALB/c mice. Similarly, priming of mice with leishmanial Ag-pulsed DCs expressing high levels of CD40 induced host protection against L. donovani challenge infection. In contrast, priming with the low CD40-expressing DC resulted in aggravated infection as compared with the control mice. The results establish that CD40 can play differential roles in Treg differentiation and determine the course of infection. We demonstrate that the knowledge can be efficiently used in adoptive cell transfer therapy against an infectious disease.

CD40 signaling in CD8+CD40+ T cells turns on contra-T regulatory cell functions.

CD4(+) regulatory T cells (Treg cells) mediate immunosuppression, whereas CD8(+) T cells confer resistance in many diseases. It is unknown whether CD8(+) T cells confer protection by antagonizing the Treg cells. Using a model of stage-specific immune responses against Leishmania donovani infection in susceptible BALB/c mice, we report that CD3(+)CD8(+)CD40(+) T cells executed CD40-dependent cytotoxicity on CD3(+)CD4(+)CD127(dim)GITR(+)CD25(+) Treg cells during the initial phase of the infection but were later apoptosed by IL-10. CD40 signaled through Ras, PI3K, and protein kinase C, resulting in p38MAPK- or ERK-1/2-independent, but NF-kappaB-dependent, induction of the cytotoxic mediators granzyme and perforin. Adoptive transfer of CD3(+)CD8(+)CD40(+) T cells reduced the L. donovani infection in BALB/c mice. These results identify CD3(+)CD8(+)CD40(+) T cells as the contra-Treg cells and imply a novel immunotherapeutic principle.

Topoisomerase inhibitors modulate expression of melanocytic antigens and enhance T cell recognition of tumor cells.

While there are many obstacles to immune destruction of autologous tumors, there is mounting evidence that tumor antigen recognition does occur. Unfortunately, immune recognition rarely controls clinically significant tumors. Even the most effective immune response will fail if tumors fail to express target antigens. Importantly, reduced tumor antigen expression often results from changes in gene regulation rather than irrevocable loss of genetic information. Such perturbations are often reversible by specific compounds or biological mediators, prompting a search for agents with improved antigen-enhancing properties. Some recent findings have suggested that certain conventional chemotherapeutic agents may have beneficial properties for cancer treatment beyond their direct cytotoxicities against tumor cells. Accordingly, we screened an important subset of these agents, topoisomerase inhibitors, for their effects on antigen levels in tumor cells. Our analyses demonstrate upregulation of antigen expression in a variety of melanoma cell lines and gliomas in response to nanomolar levels of certain specific topoisomerase inhibitors. To demonstrate the ability of CD8+ T cells to recognize tumors, we assayed cytokine secretion in T cells transfected with T cell receptors directed against Melan-A/MART-1 antigen. Three days of daunorubicin treatment resulted in enhanced antigen expression by tumor cells, in turn inducing co-cultured antigen-specific T cells to secrete Interleukin-2 and Interferon-γ. These results demonstrate that specific topoisomerase inhibitors can augment melanoma antigen production, suggesting that a combination of chemotherapy and immunotherapy may be of potential value in the treatment of otherwise insensitive cancers.

Miltefosine promotes IFN-gamma-dominated anti-leishmanial immune response.

Leishmania donovani, a protozoan parasite, resides and replicates as amastigotes within macrophages. The parasite inflicts the disease visceral leishmaniasis by suppressing host cell function. Neither a therapeutic vaccine nor an effective anti-leishmanial drug to reverse the immunosuppression is available. Although miltefosine (hexadecylphosphocholine or HPC) is a promising orally bioavailable anti-leishmanial drug, its efficacy is seriously compromised by contra-indications in pregnant women. Further rational redesigning of the drug requires studies on its mechanism of action, which is unknown at present. Because miltefosine is proposed to have immunomodulatory functions, we examined whether miltefosine exerts its anti-leishmanial functions by activating macrophages. We observed that miltefosine's anti-leishmanial function was significantly compromised in IFN-gamma-deficient macrophages suggesting the importance of endogenous IFN-gamma in miltefosine-induced anti-leishmanial functions of macrophages. Miltefosine induced IFN-gamma, neutralization of which reduced the anti-leishmanial functions of macrophages. IFN-gamma responsiveness is reduced in L. donovani-infected macrophages but is significantly restored by miltefosine, as it enhances IFN-gamma receptors and IFN-gamma induced STAT-1 phosphorylation but reduced activation of SHP-1, the phosphatase implicated in the down-regulation of STAT-1 phosphorylation. Miltefosine induced protein kinase C-dependent and PI3K-dependent p38MAP kinase phosphorylation and anti-leishmanial function. Miltefosine promotes p38MAP kinase-dependent anti-leishmanial functions and IL-12-dependent Th1 response. Leishmania donovani-infected macrophages induced Th2 response but miltefosine treatment reversed the response to Th1-type. Thus, our data define for the first time the mechanistic basis of host cell-dependent anti-leishmanial function of miltefosine.

SEB-induced signaling in macrophages leads to biphasic TNF-alpha.

APCs express MHC-II molecules. Binding of enterotoxins to MHC-II generates a signal resulting in the production of TNF-alpha that mediates toxic shock syndrome. However, the signaling events that lead to TNF-alpha production in macrophages are not well understood. We, for the first time, demonstrate that binding of staphylococcal enterotoxin B to MHC-II results in activation of TNF-alpha-converting enzyme, epidermal growth factor receptor, p38MAPK, and NF-kappaB inducing biphasic TNF-alpha production. Paraformaldehyde-fixed, peptide-specific T cells also activate MHC-II signaling and TNF-alpha induction in peptide-pulsed macrophages. Our results reveal a novel MHC-II signaling and bidirectional macrophage-T cell interaction regulating macrophage functions. This knowledge may help to develop novel, macrophage-directed, therapeutic strategies.

The human CD8ß M-4 isoform dominant in effector memory T cells has distinct cytoplasmic motifs that confer unique properties.

The CD8 co-receptor influences T cell recognition and responses in both anti-tumor and anti-viral immunity. During evolution in the ancestor of humans and chimpanzees, the CD8B gene acquired two additional exons. As a result, in humans, there are four CD8ß splice variants (M1 to M4) that differ in their cytoplasmic tails. The M-1 isoform which is the equivalent of murine CD8ß, is predominantly expressed in naïve T cells, whereas, the M-4 isoform is predominantly expressed in effector memory T cells. The characteristics of the M-4 isoform conferred by its unique 36 amino acid cytoplasmic tail are not known. In this study, we identified a dihydrophobic leucine-based receptor internalization motif in the cytoplasmic tail of M-4 that regulated its cell surface expression and downregulation after activation. Further the M-4 cytoplasmic tail was able to associate with ubiquitinated targets in 293T cells and mutations in the amino acids NPW, a potential EH domain binding site, either enhanced or inhibited the interaction. In addition, the M-4 tail was itself mono-ubiquitinated on a lysine residue in both 293T cells and a human T cell line. When peripheral blood human T cells expressed CD8αß M-4, the frequency of MIP-1ß secreting cells responding to antigen presenting cells was two-fold higher as compared to CD8αß M-1 expressing T cells. Thus, the cytoplasmic tail of the CD8ß M-4 isoform has unique characteristics, which likely contributed to its selective expression and function in human effector memory T cells.

CD25 blockade depletes and selectively reprograms regulatory T cells in concert with immunotherapy in cancer patients.

Regulatory T cells (T(regs)) are key mediators of immune tolerance and feature prominently in cancer. Depletion of CD25(+) FoxP3(+) T(regs) in vivo may promote T cell cancer immunosurveillance, but no strategy to do so in humans while preserving immunity and preventing autoimmunity has been validated. We evaluated the Food and Drug Administration-approved CD25-blocking monoclonal antibody daclizumab with regard to human T(reg) survival and function. In vitro, daclizumab did not mediate antibody-dependent or complement-mediated cytotoxicity but rather resulted in the down-regulation of FoxP3 selectively among CD25(high) CD45RA(neg) T(regs). Moreover, daclizumab-treated CD45RA(neg) T(regs) lost suppressive function and regained the ability to produce interferon-γ, consistent with reprogramming. To understand the impact of daclizumab on T(regs) in vivo, we performed a clinical trial of daclizumab in combination with an experimental cancer vaccine in patients with metastatic breast cancer. Daclizumab administration led to a marked and prolonged decrease in T(regs) in patients. Robust CD8 and CD4 T cell priming and boosting to all vaccine antigens were observed in the absence of autoimmunity. We conclude that CD25 blockade depletes and selectively reprograms T(regs) in concert with active immune therapy in cancer patients. These results suggest a mechanism to target cancer-associated T(regs) while avoiding autoimmunity.

CD40-induced countercurrent conduits for tumor escape or elimination?

CD40--which is expressed on endothelial cells and antigen-presenting cells, such as B cells, macrophages and dendritic cells--is a glycoprotein receptor for T cell-expressed CD40-ligand. CD40 signaling leads to production of both pro-inflammatory and anti-inflammatory mediators. Some of these mediators, such as vascular endothelial growth factor, help in angiogenesis, forming new capillaries. Although these capillaries can function as conduits for tumor-cell metastasis, these same vessels can also be a gateway for entry of activated anti-tumor effector cells that eliminate tumors. How such countercurrent processes can be turned in favor of anti-tumor immunity remains to be shown.

Inhibition of IL-2 induced IL-10 production as a principle of phase-specific immunotherapy.

Leishmania donovani, a protozoan parasite, inflicts a fatal disease, visceral leishmaniasis. The suppression of antileishmanial T cell responses that characterizes the disease was proposed to be due to deficiency of a T cell growth factor, IL-2. We demonstrate that during the first week after L. donovani infection, IL-2 induces IL-10 that suppresses the host-protective functions of T cells 14 days after infection. The observed suppression is concurrent with increased CD4+ glucocorticoid-induced TNF receptor+ T cells and Foxp3 expression in BALB/c mice, implicating IL-2-dependent regulatory T cell control of antileishmanial immune responses. Indeed, IL-2 and IL-10 neutralization at different time points after the infection demonstrates their distinct roles at the priming and effector phases, respectively, and establishes kinetic modulation of ongoing immune responses as a principle of a rational, phase-specific immunotherapy.