Evaluation of the Immune Response within the Tumor Microenvironment in African American and non-Hispanic White Non-Small Cell Lung Cancer Patients.

BACKGROUND: African Americans have higher incidence and mortality from lung cancer than non-Hispanic Whites, but investigations into differences in immune response have been minimal. Therefore, we compared components of the tumor microenvironment among African Americans and non-Hispanic Whites diagnosed with non-small cell lung cancer (NSCLC) based on PD-L1 or tertiary lymphoid structure (TLS) status to identify differences of translational relevance. METHODS: Using a cohort of 280 NSCLC patients from the INHALE study (non-Hispanic White: n=155; African American: n=125), we evaluated PD-L1 tumor proportion score (<1% vs. ≥1%) and TLS status (presence/absence), comparing differences within the tumor microenvironment based on immune cell distribution and differential expression of genes. RESULTS: Tumors from African Americans had a higher proportion of plasma cell signatures within the tumor microenvironment than non-Hispanic Whites. In addition, gene expression patterns in African American PD-L1 positive samples suggest these tumors contained greater numbers of γδ T-cells and resting dendritic cells, along with fewer CD8+ T-cells after adjusting for age, sex, pack-years, stage, and histology. Investigation of differential expression of B-cell/plasma cell related genes between the two patient populations revealed that two immunoglobulin genes (IGKV2-29 and IGLL5) were associated with decreased mortality risk in African Americans. CONCLUSIONS: In the first known race-stratified analysis of tumor microenvironment components in lung cancer based on PD-L1 expression or TLS status, differences within the immune cell composition and transcriptomic signature were identified that may have therapeutic implications. IMPACT: Future investigation of racial variation within the tumor microenvironment may help direct the use of immunotherapy.

Interferon gamma immunoPET imaging to evaluate response to immune checkpoint inhibitors.

Introduction: We previously developed a 89Zr-labeled antibody-based immuno-positron emission tomography (immunoPET) tracer targeting interferon gamma (IFNγ), a cytokine produced predominantly by activated T and natural killer (NK) cells during pathogen clearance, anti-tumor immunity, and various inflammatory and autoimmune conditions. The current study investigated [89Zr]Zr-DFO-anti-IFNγ PET as a method to monitor response to immune checkpoint inhibitors (ICIs). Methods: BALB/c mice bearing CT26 colorectal tumors were treated with combined ICI (anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and anti-programmed death 1 (PD-1)). The [89Zr]Zr-DFO-anti-IFNγ PET tracer, generated with antibody clone AN18, was administered on the day of the second ICI treatment, with PET imaging 72 hours later. Tumor mRNA was analyzed by quantitative reverse-transcribed PCR (qRT-PCR). Results: We detected significantly higher intratumoral localization of [89Zr]Zr-DFO-anti-IFNγ in ICI-treated mice compared to untreated controls, while uptake of an isotype control tracer remained similar between treated and untreated mice. Interestingly, [89Zr]Zr-DFO-anti-IFNγ uptake was also elevated relative to the isotype control in untreated mice, suggesting that the IFNγ-specific tracer might be able to detect underlying immune activity in situ in this immunogenic model. In an efficacy experiment, a significant inverse correlation between tracer uptake and tumor burden was also observed. Because antibodies to cytokines often exhibit neutralizing effects which might alter cellular communication within the tumor microenvironment, we also evaluated the impact of AN18 on downstream IFNγ signaling and ICI outcomes. Tumor transcript analysis using interferon regulatory factor 1 (IRF1) expression as a readout of IFNγ signaling suggested there may be a marginal disruption of this pathway. However, compared to a 250 µg dose known to neutralize IFNγ, which diminished ICI efficacy, a tracer-equivalent 50 µg dose did not reduce ICI response rates. Discussion: These results support the use of IFNγ PET as a method to monitor immune activity in situ after ICI, which may also extend to additional T cell-activating immunotherapies.

Host genetic background regulates the capacity for anti-tumor antibody-dependent phagocytosis.

Background: Antitumor antibody, or targeted immunotherapy, has revolutionized cancer treatment and markedly improved patient outcomes. A prime example is the monoclonal antibody (mAb) trastuzumab, which targets human epidermal growth factor receptor 2 (HER2). However, like many targeted immunotherapies, only a subset of patients benefit from trastuzumab long-term. In addition to tumor-intrinsic factors, we hypothesize that host genetics may influence subsequent immune activation. Methods: To model the human population, we produced F1 crosses of genetically heterogeneous Diversity Outbred (DO) mice with BALB/c mice (DOCF1). Distinct DOCF1 mice were orthotopically implanted with the BALB/c-syngeneic TUBO mammary tumor line, which expresses the HER2 ortholog rat neu. Treatment with anti-neu mAb clone 7.16.4 began once tumors reached ∼200 mm 3 . Genetic linkage and quantitative trait locus (QTL) effects analyses in R/qtl2 identified loci associated with tumor growth rates. Locus validation was performed with BALB/c F1 crosses with recombinant-inbred Collaborative Cross (CC) strains selected for therapy-associated driver genetics (CCxCF1). The respective roles of natural killer (NK) cells and macrophages were investigated by selective depletion in vivo. Ex vivo macrophage antibody-dependent phagocytosis (ADCP) assays were evaluated by confocal microscopy using 7.16.4-opsonized E2Crimson-expressing TUBO tumor cells. Results: We observed a divergent response to anti-tumor antibody therapy in DOCF1 mice. Genetic linkage analysis detected a locus on chromosome 10 that correlates to a robust response to therapy, which was validated in CCxCF1 models. Single-cell RNA sequencing of tumors from responder and non-responder models identified key differences in tumor immune infiltrate composition, particularly within macrophage (Mφ) subsets. This is further supported by ex vivo analysis showing Mφ ADCP capacity correlates to in vivo treatment outcomes in both DOCF1 and CCxCF1 models. Conclusions: Host genetics play a key regulatory role in targeted immunotherapy outcomes, and putative causal genes are identified in murine chromosome 10 which may govern Mφ function during ADCP.

Protocolized Training of Advanced Practice Providers for Robotic Surgery Improves the Quality of Intraoperative Assistance.

BACKGROUND: The expansion of robotic surgery requires identifying factors of competent robotic bedside assisting. Surgical trainees desire more robotic console time, and we hypothesized that protocolized robotic surgery bedside training could equip Advanced Practice Providers (APPs) to meet this growing need. No standardized precedent exists for training APPs. METHODS: We designed a pilot study consisting of didactic and clinical skills. APPs completed didactic tests followed by proctored clinical skills checklists intraoperatively. Operating surgeons scored trainees with 10-point Likert scale (< 5 not confident, > 5 = confident). APPs scoring > 5 advanced to a solo practicum. Competence was defined as: didactic test score > 75th percentile, completing < 5 checklists, scoring > 5 on the practicum. The probability of passing the practicum was calculated with Bayes theorem. RESULTS: Of 10 APP trainees, 5 passed on initial attempt. After individualized development plans, 4 passed retesting. Differences in trainee factors were not statistically significant, but the probability of passing the practicum was < 50% if more than four checklists were needed. CONCLUSIONS: Clinical experience, not didactic knowledge, determines the probability of intraoperative competence. Increasing clinical proctoring did not result in higher probability of competence. Early identification of APPs needing individualized improvement increases the proportion of competent APPs.

Evaluation of an ImmunoPET Tracer for IL-12 in a Preclinical Model of Inflammatory Immune Responses.

The immune cytokine interleukin-12 (IL-12) is involved in cancer initiation and progression, autoimmunity, as well as graft versus host disease. The ability to monitor IL-12 via imaging may provide insight into various immune processes, including levels of antitumor immunity, inflammation, and infection due to its functions in immune signaling. Here, we report the development and preclinical evaluation of an antibody-based IL-12-specific positron emission tomography (PET) tracer. To mimic localized infection and stimulate IL-12 production, BALB/c mice were administered lipopolysaccharide (LPS) intramuscularly. [89Zr]Zr-DFO-αIL12 tracer was given one hour post LPS administration and PET images were taken after 5, 24, 48, and 72 hours. We observed significantly higher uptake in LPS-treated mice as compared to controls. Biodistribution of the tracer was evaluated in a separate cohort of mice, where tracer uptake was elevated in muscle, spleen, lymph nodes, and intestines after LPS administration. To evaluate the utility of [89Zr]Zr-DFO-αIL12 as an indicator of antigen presenting cell activation after cancer immunotherapy, we compared PET imaging with and without intratumoral delivery of oncolytic adenovirus expressing granulocyte-macrophage colony-stimulating factor (Adv/GM-CSF), which we have shown promotes anti-tumor immunity. BALB/c mice were inoculated orthotopically with the mouse mammary carcinoma line TUBO. Once TUBO tumors reached a volume of ~50 mm3, mice were treated with either three intratumoral injections of 108 PFU Adv/GM-CSF or vehicle control, given every other day. Upon the last dose, [89Zr]Zr-DFO-αIL12 was injected intravenously and 72 hours later all mice were imaged via PET. Tumor-specific uptake of [89Zr]Zr-DFO-αIL12 was higher in Adv/GM-CSF treated mice versus controls. Tissues were harvested after imaging, and elevated levels of macrophages and CD8+ Tc cells were detected in Adv/GM-CSF treated tumors by immunohistochemistry. We validated that IL-12 expression was induced after Adv/GM-CSF by qRT-PCR. Importantly, expression of genes activated by IL-12 (IFNγ, TNFα, and IL-18) were unaffected after IL-12 imaging relative to mice receiving an IgG control tracer, suggesting the tracer antibody does not significantly disrupt signaling. Our results indicate that targeting soluble cytokines such as IL-12 by PET imaging with antibody tracers may serve as a noninvasive method to evaluate the function of the immune milieu in situ.

A diversity outbred F1 mouse model identifies host-intrinsic genetic regulators of response to immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICI) have improved outcomes for a variety of malignancies; however, many patients fail to benefit. While tumor-intrinsic mechanisms are likely involved in therapy resistance, it is unclear to what extent host genetic background influences response. To investigate this, we utilized the Diversity Outbred (DO) and Collaborative Cross (CC) mouse models. DO mice are an outbred stock generated by crossbreeding eight inbred founder strains, and CC mice are recombinant inbred mice generated from the same eight founders. We generated 207 DOB6F1 mice representing 48 DO dams and demonstrated that these mice reliably accept the C57BL/6-syngeneic B16F0 tumor and that host genetic background influences response to ICI. Genetic linkage analysis from 142 mice identified multiple regions including one within chromosome 13 that associated with therapeutic response. We utilized 6 CC strains bearing the positive (NZO) or negative (C57BL/6) driver genotype in this locus. We found that 2/3 of predicted responder CCB6F1 crosses show reproducible ICI response. The chromosome 13 locus contains the murine prolactin family, which is a known immunomodulating cytokine associated with various autoimmune disorders. To directly test whether prolactin influences ICI response rates, we implanted inbred C57BL/6 mice with subcutaneous slow-release prolactin pellets to induce mild hyperprolactinemia. Prolactin augmented ICI response against B16F0, with increased CD8 infiltration and 5/8 mice exhibiting slowed tumor growth relative to controls. This study highlights the role of host genetics in ICI response and supports the use of F1 crosses in the DO and CC mouse populations as powerful cancer immunotherapy models.

The current state of robotic retromuscular repairs-a qualitative review of the literature.

BACKGROUND: The management of ventral incisional hernias (VIH) has undergone many iterations over the last 5 years due to evolution in surgical techniques and advancement in robotic surgery. Four general principles have emerged: mesh usage, retromuscular mesh placement, primary fascial closure, and usage of minimally invasive techniques when possible. The application of robotic retromuscular repairs in VIH allow these principles to be applied simultaneously. This qualitative review attempts to answer what robotic retromuscular repairs are described, which patients are selected for these techniques, and what are current outcomes. METHODS: Using the key words: "robotic retromuscular repair", "robotic Rives Stoppa", and "robotic transversus abdominis release", a PubMed search of articles written up to December 2019 was critically reviewed. RESULTS: 44 articles were encountered, 9 high-quality articles were analyzed for this manuscript. Level of evidence ranged from 2B to 2C. Robotic TAR patients had BMI of 33 kg/m2, defect sizes ranging from 7-14 cm wide to 12-19 cm long, longer OR times, no difference in surgical site events, and shorter length of stay (LOS). The techniques to perform robotic Rives Stoppa (RS) were heterogeneous; however, extended totally extraperitoneal (ETEP) approach is most described. Defect width for RS repairs ranged 4-7 cm and LOS was less than 1 day. Complication rates were low, there is no long-term data on hernia recurrence, and information on cost is limited. CONCLUSION: In short-term follow-up, robotic retromuscular repairs show promise that VIH can be repaired with intramuscular mesh, few complications, and shorter LOS. Data on hernia recurrence, long-term complications, and rigorous cost analysis are needed to demonstrate generalizability.

Diversity Outbred Mice Reveal the Quantitative Trait Locus and Regulatory Cells of HER2 Immunity.

The genetic basis and mechanisms of disparate antitumor immune response was investigated in Diversity Outbred (DO) F1 mice that express human HER2. DO mouse stock samples nearly the entire genetic repertoire of the species. We crossed DO mice with syngeneic HER2 transgenic mice to study the genetics of an anti-self HER2 response in a healthy outbred population. Anti-HER2 IgG was induced by Ad/E2TM or naked pE2TM, both encoding HER2 extracellular and transmembrane domains. The response of DO F1 HER2 transgenic mice was remarkably variable. Still, immune sera inhibited HER2+ SKBR3 cell survival in a dose-dependent fashion. Using DO quantitative trait locus (QTL) analysis, we mapped the QTL that influences both total IgG and IgG2(a/b/c) Ab response to either Ad/E2TM or pE2TM. QTL from these four datasets identified a region in chromosome 17 that was responsible for regulating the response. A/J and NOD segments of genes in this region drove elevated HER2 Ig levels. This region is rich in MHC-IB genes, several of which interact with inhibitory receptors of NK cells. (B6xA/J)F1 and (B6xNOD)F1 HER2 transgenic mice received Ad/E2TM after NK cell depletion, and they produced less HER2 IgG, demonstrating positive regulatory function of NK cells. Depletion of regulatory T cells enhanced response. Using DO QTL analysis, we show that MHC-IB reactive NK cells exert positive influence on the immunity, countering negative regulation by regulatory T cells. This new, to our knowledge, DO F1 platform is a powerful tool for revealing novel immune regulatory mechanisms and for testing new interventional strategies.

In vivo Imaging Technologies to Monitor the Immune System.

The past two decades have brought impressive advancements in immune modulation, particularly with the advent of both cancer immunotherapy and biologic therapeutics for inflammatory conditions. However, the dynamic nature of the immune response often complicates the assessment of therapeutic outcomes. Innovative imaging technologies are designed to bridge this gap and allow non-invasive visualization of immune cell presence and/or function in real time. A variety of anatomical and molecular imaging modalities have been applied for this purpose, with each option providing specific advantages and drawbacks. Anatomical methods including magnetic resonance imaging (MRI), computed tomography (CT), and ultrasound provide sharp tissue resolution, which can be further enhanced with contrast agents, including super paramagnetic ions (for MRI) or nanobubbles (for ultrasound). Conjugation of the contrast material to an antibody allows for specific targeting of a cell population or protein of interest. Protein platforms including antibodies, cytokines, and receptor ligands are also popular choices as molecular imaging agents for positron emission tomography (PET), single-photon emission computerized tomography (SPECT), scintigraphy, and optical imaging. These tracers are tagged with either a radioisotope or fluorescent molecule for detection of the target. During the design process for immune-monitoring imaging tracers, it is important to consider any potential downstream physiologic impact. Antibodies may deplete the target cell population, trigger or inhibit receptor signaling, or neutralize the normal function(s) of soluble proteins. Alternatively, the use of cytokines or other ligands as tracers may stimulate their respective signaling pathways, even in low concentrations. As in vivo immune imaging is still in its infancy, this review aims to describe the modalities and immunologic targets that have thus far been explored, with the goal of promoting and guiding the future development and application of novel imaging technologies.

Removal of Fc Glycans from [89Zr]Zr-DFO-Anti-CD8 Prevents Peripheral Depletion of CD8+ T Cells.

The N-linked biantennary glycans on the heavy chain of immunoglobulin G (IgG) antibodies (mAbs) are instrumental in the recognition of the Fc region by Fc-gamma receptors (FcγR). In the case of full-length mAb-based imaging tracers targeting immune cell populations, these Fc:FcγR interactions can potentially deplete effector cells responsible for tumor clearance. To bypass this problem, we hypothesize that the enzymatic removal of the Fc glycans will disrupt Fc:FcγR interactions and spare tracer-targeted immune cells from depletion during immunopositron emission tomography (immunoPET) imaging. Herein, we compared the in vitro and in vivo properties of 89Zr-radiolabeled CD8-specific murine mAb (anti-CD8wt, clone 2.43), a well-known depleting mAb, and its deglycosylated counterpart (anti-CD8degly). Deglycosylation was achieved via enzymatic treatment with the peptide: N-glycosidase F (PNGaseF). Both anti-CD8wt and anti-CD8degly mAbs were conjugated to p-SCN-Bn-desferrioxamine (DFO) and labeled with 89Zr. Bindings of both DFO-conjugated mAbs to FcγR and CD8+ splenocytes were compared. In vivo imaging and distribution studies were conducted to examine the specificity and pharmacokinetics of the radioimmunoconjugates in tumor-naive and CT26 colorectal tumor-bearing mice. Ex vivo analysis of CD8+ T cell population in spleens and tumors obtained postimaging were measured via flow cytometry and qRT-PCR. The removal of the Fc glycans from anti-CD8wt was confirmed via SDS-PAGE. A reduction in FcγR interaction was exhibited by DFO-anti-CD8degly, while its binding to CD8 remained unchanged. Tissue distribution showed similar pharmacokinetics of [89Zr]Zr-DFO-anti-CD8degly and the wt radioimmunoconjugate. In vivo blocking studies further demonstrated retained specificity of the deglycosylated radiotracer for CD8. From the imaging studies, no difference in accumulation in both spleens and tumors was observed between both radiotracers. Results from the flow cytometry analysis confirmed depletion of CD8+ T cells in spleens of mice administered with DFO-anti-CD8wt, whereas an increase in CD8+ T cells was shown with DFO-anti-CD8degly. No statistically significant difference in tumor infiltrating CD8+ T cells was observed in cohorts administered with the probes when compared to control unmodulated mice. CD8 mRNA levels from excised tumors showed increased transcripts of the antigen in mice administered with [89Zr]Zr-DFO-anti-CD8degly compared to mice imaged with [89Zr]Zr-DFO-anti-CD8wt. In conclusion, the removal of Fc glycans offers a straightforward approach to develop full length antibody-based imaging probes specifically for detecting CD8+ immune molecules with no consequential depletion of their target cell population in peripheral tissues.

An HER2 DNA vaccine with evolution-selected amino acid substitutions reveals a fundamental principle for cancer vaccine formulation in HER2 transgenic mice.

Enhancement of endogenous immunity to tumor-associated self-antigens and neoantigens is the goal of preventive vaccination. Toward this goal, we compared the efficacy of the following HER2 DNA vaccine constructs: vaccines encoding wild-type HER2, hybrid HER2 vaccines consisting of human HER2 and rat Neu, HER2 vaccines with single residue substitutions and a novel human HER2 DNA vaccine, ph(es)E2TM. ph(es)E2TM was designed to contain five evolution-selected substitutions: M198V, Q398R, F425L, H473R and A622T that occur frequently in 12 primate HER2 sequences. These ph(es)E2TM substitutions score 0 to 1 in blocks substitutions matrix (BLOSUM), indicating minimal biochemical alterations. h(es)E2TM recombinant protein is recognized by a panel of anti-HER2 mAbs, demonstrating the preservation of HER2 protein structure. Compared to native human HER2, electrovaccination of HER2 transgenic mice with ph(es)E2TM induced a threefold increase in HER2-binding antibody (Ab) and elevated levels of IFNγ-producing T cells. ph(es)E2TM, but not pE2TM immune serum, recognized HER2 peptide p95 355LPESFDGDPASNTAP369, suggesting a broadening of epitope recognition induced by the minimally modified HER2 vaccine. ph(es)E2TM vaccination reduced tumor growth more effectively than wild-type HER2 or HER2 vaccines with more extensive modifications. The elevation of tumor immunity by ph(es)E2TM vaccination would create a favorable tumor microenvironment for neoantigen priming, further enhancing the protective immunity. The fundamental principle of exploiting evolution-selected amino acid substitutions is novel, effective and applicable to vaccine development in general.

IFNγ PET Imaging as a Predictive Tool for Monitoring Response to Tumor Immunotherapy.

IFNγ is an attractive target for imaging active antitumor immunity due to its function in the T-cell signaling axis. Here, we test an IFNγ immuno-PET (immunoPET) probe for its capacity to identify adaptive immunotherapy response after HER2/neu vaccination in both spontaneous salivary and orthotopic neu+ mouse mammary tumors. IFNγ immunoPET detected elevated cytokine levels in situ after vaccination, which inversely correlated with tumor growth rate, an indicator of response to therapy. In a model of induced T-cell anergy where CD8 T cells infiltrate the tumor, but upregulate PD-1, IFNγ tracer uptake was equivalent to isotype control, illustrating a lack of antitumor T-cell activity. The IFNγ immunoPET tracer detected IFNγ protein sequestered on the surface of tumor cells, likely in complex with the IFNγ receptor, which may explain imaging localization of this soluble factor in vivo Collectively, we find that the activation status of cytotoxic T cells is annotated by IFNγ immunoPET, with reduced off-target binding to secondary lymphoid tissues compared with imaging total CD3+ tumor-infiltrating lymphocytes. Targeting of soluble cytokines such as IFNγ by PET imaging may provide valuable noninvasive insight into the function of immune cells in situ Significance: This study presents a novel approach to monitor therapeutic outcomes via IFNγ-targeted positron emission tomography. Cancer Res; 78(19); 5706-17. ©2018 AACR.

In situ immunization via non-surgical ablation to prevent local and distant tumor recurrence.

Host immunities are induced during cryoablation or oncolytic adenovirus therapy when the entire repertoire of tumor associated antigens (TAA) is released. Local and systemic protection is enhanced by the combined treatment with toll-like receptor agonist or immune stimulating cytokines. Non-surgical tumor ablation is an effective platform for in situ immunization.

Induction of HER2 Immunity in Outbred Domestic Cats by DNA Electrovaccination.

Domestic cats share human living environments and genetic traits. They develop spontaneous feline mammary carcinoma (FMC) with similar histopathology to human breast cancer. HER2 and AKT phosphorylation was demonstrated in primary FMC by immunoblot analysis, indicating HER2 as a therapeutic target. FMC lines K12 and K248 expressing HER1, HER2, and HER3 were sensitive to receptor tyrosine kinase (RTK) inhibitors gefitinib and lapatinib. To test HER2 vaccine response in cats, purpose-bred, healthy cats were electrovaccinated with heterologous (xenogeneic) or point-mutated feline HER2 DNA. T-cell reactivity to feline self-HER2 was detected in 4 of 10 cats that received bear HER2, human-rat fusion HER2 (E2Neu) or mutant feline HER2 (feHER2-K), which contains a single amino acid substitution. The variable T-cell responses may resemble that in the genetically heterogeneous human population. All immune sera to heterologous HER2 recognized feline HER2 expressed in 3T3 cells (3T3/HER2), but not that in FMC K12 or K248. Immune sera to mutant pfeHER2-K bound 3T3/HER2 cells weakly, but they showed better recognition of K12 and K248 cells that also express HER1 and HER3, suggesting distinct HER2 epitopes displayed by FMC that may be simulated by feHER2-K. In summary, HER2 DNA electroporation overcomes T-cell immune tolerance in approximately 40% of healthy cats and induces antibodies with distinct specificity. Vaccination studies in domestic cats can expedite vaccine iteration to guide human vaccine design and better predict outcome, with the added benefit of helping feline mammary tumor patients.

Cryotherapy with concurrent CpG oligonucleotide treatment controls local tumor recurrence and modulates HER2/neu immunity.

Percutaneous cryoablation is a minimally invasive procedure for tumor destruction, which can potentially initiate or amplify antitumor immunity through the release of tumor-associated antigens. However, clinically efficacious immunity is lacking and regional recurrences are a limiting factor relative to surgical excision. To understand the mechanism of immune activation by cryoablation, comprehensive analyses of innate immunity and HER2/neu humoral and cellular immunity following cryoablation with or without peritumoral CpG injection were conducted using two HER2/neu(+) tumor systems in wild-type (WT), neu-tolerant, and SCID mice. Cryoablation of neu(+) TUBO tumor in BALB/c mice resulted in systemic immune priming, but not in neu-tolerant BALB NeuT mice. Cryoablation of human HER2(+) D2F2/E2 tumor enabled the functionality of tumor-induced immunity, but secondary tumors were refractory to antitumor immunity if rechallenge occurred during the resolution phase of the cryoablated tumor. A step-wise increase in local recurrence was observed in WT, neu-tolerant, and SCID mice, indicating a role of adaptive immunity in controlling residual tumor foci. Importantly, local recurrences were eliminated or greatly reduced in WT, neu tolerant, and SCID mice when CpG was incorporated in the cryoablation regimen, showing significant local control by innate immunity. For long-term protection, however, adaptive immunity was required because most SCID mice eventually succumbed to local tumor recurrence even with combined cryoablation and CpG treatment. This improved understanding of the mechanisms by which cryoablation affects innate and adaptive immunity will help guide appropriate combination of therapeutic interventions to improve treatment outcomes.

Impaired proteasome function activates GATA3 in T cells and upregulates CTLA-4: relevance for Sézary syndrome.

Highly regulated expression of the negative costimulatory molecule cytotoxic T-lymphocyte antigen-4 (CTLA-4) on T cells modulates T-cell activation and proliferation. CTLA-4 is preferentially expressed in Th2 T cells, whose differentiation depends on the transcriptional regulator GATA3. Sézary syndrome (SS) is a T-cell malignancy characterized by Th2 cytokine skewing, impaired T-cell responses, and overexpression of GATA3 and CTLA-4. GATA3 is regulated by phosphorylation and ubiquitination. In SS cells, we detected increased polyubiquitinated proteins and activated GATA3. We hypothesized that proteasome dysfunction in SS T cells may lead to GATA3 and CTLA-4 overexpression. To test this hypothesis, we blocked proteasome function with bortezomib in normal T cells, and observed sustained GATA3 and CTLA-4 upregulation. The increased CTLA-4 was functionally inhibitory in a mixed lymphocyte reaction (MLR). GATA3 directly transactivated the CTLA-4 promoter, and knockdown of GATA3 messenger RNA and protein inhibited CTLA-4 induction mediated by bortezomib. Finally, knockdown of GATA3 in patient's malignant T cells suppressed CTLA-4 expression. Here we demonstrate a new T-cell regulatory pathway that directly links decreased proteasome degradation of GATA3, CTLA-4 upregulation, and inhibition of T-cell responses. We also demonstrate the requirement of the GATA3/CTLA-4 regulatory pathway in fresh neoplastic CD4+ T cells. Targeting of this pathway may be beneficial in SS and other CTLA-4-overexpressing T-cell neoplasms.

Immune function abnormalities in peripheral blood mononuclear cell cytokine expression differentiates stages of cutaneous T-cell lymphoma/mycosis fungoides.

PURPOSE: Mycosis fungoides (MF) is a cutaneous T-cell lymphoma (CTCL) characterized by neoplastic skin-homing T cells. To better understand the immunopathogenesis of MF, we analyzed the functional ability of peripheral blood mononuclear cells (PBMC) from early and late MF/CTCL patients to express cytokine genes. In late stage MF/CTCL, patients were separated into those with blood involvement (+B) and without blood involvement (-B). EXPERIMENTAL DESIGN: We analyzed T(H)1 (interleukin 2 (IL-2), IFN-gamma), T(H)2 (IL-4, IL-5, IL-10, IL-13), and T(H)17 (IL-17) cytokine gene expression from activated PBMCs from normal (n = 12), psoriasis (n = 6), early MF/CTCL (n = 11), and late MF/CTCL+B (n = 4) and MF/CTCL-B (n = 3) by quantitative real-time PCR. RESULTS: PBMCs from early MF/CTCL and psoriasis showed higher induction of IL-2, IL-4, and IFN-gamma genes than those from normal and late MF/CTCL-B and MF/CTCL+B (P < 0.05) in descending order. PBMCs from late MF/CTCL-B exhibited generally the highest level of IL-5, IL-10, IL-13, and IL-17 expression compared with the other groups. PBMCs from early MF/CTCL and late MF/CTCL-B had similarly elevated IL-13 and IL-17. Of all groups, PBMCs from late MF/CTCL+B had the lowest levels of IL-2 (P < 0.05), IL-4, IFN-gamma, IL-13, and IL-17. CONCLUSIONS: The different pattern of cytokine gene expression suggests a change in immune function in MF/CTCL from early MF/CTCL to late MF/CTCL-B to late MF/CTCL+B. These stages are consistent with localized disease associated with an anti-tumor immune response and late MF/CTCL associated with a loss of immune function mediated by malignant T cells that share regulatory T cell-like properties.

Induction of the CTLA-4 gene in human lymphocytes is dependent on NFAT binding the proximal promoter.

CTLA-4 is a member of the costimulatory family, has homology to CD28, and binds the B7 family of ligands. Unlike CD28, CTLA-4 ligation transmits a negative signal in T cells. CTLA-4 expression, while inducible in most T cells, is expressed constitutively on T cells with a regulatory phenotype. The mechanism controlling CTLA-4 expression in human T cells is poorly characterized, thus we sought to better understand the mechanism of activation of the CTLA-4 gene. By cloning the 5' upstream promoter and creating promoter-deletion reporter constructs, we show that the proximal promoter is critical for activating the CTLA-4 gene. Within this region, we identify a NFAT consensus sequence that binds NFAT with high affinity that differs from other NFAT sequences and does not recruit AP-1. Analysis of the chromatin proteins in the native CTLA-4 gene shows that this promoter region becomes associated with acetylated histones by chromatin immunoprecipitation assays. In addition, NFAT1 binds to the promoter of the CTLA-4 gene after stimulation by chromatin immunoprecipitation. The functional requirement of the NFAT site for CTLA-4 transcription was demonstrated by mutations in the NFAT site that abolished the activity of the promoter. Furthermore, inhibitors of NFAT suppressed CTLA-4 gene expression, indicating that NFAT plays a critical role in regulating the induction of the CTLA-4 gene in lymphocytes. The identification of NFAT as a critical regulator of the CTLA-4 gene suggests that targeting NFAT function may lead to novel approaches to modulate the CTLA-4 gene to control the immune response.

Increased expression of CTLA-4 in malignant T-cells from patients with mycosis fungoides -- cutaneous T cell lymphoma.

Mycosis fungoides (MF) is a low-grade lymphoma of cluster of differentiation (CD)4+, CD45RO+, cutaneous leukocyte antigen (CLA)+ T cells that homes to the skin. To understand the functional abnormalities in this disease, we study the regulation of cytotoxic T-lymphocyte antigen (CTLA)-4 in peripheral blood mononuclear cells (PBMCs) from patients with MF. CTLA-4 is a costimulatory molecule for T cells that functions in immunoregulation. Unlike the expression of CD28, which is expressed constitutively on T cells, CTLA-4 expression is highly regulated. In the analysis of PBMCs in MF, we found that CTLA-4 is stimulated by phorbol myristate acetate/A23187 to a greater level when compared to normals. This defect was seen in the dominant clones of T cells. The increased CTLA-4 expression was significant between normal and MF, with a correlation between higher expression of CTLA-4 and a higher grade of MF. In a patient whose disease progressed, the CTLA-4 level increased. The abnormal level of CTLA-4 was confirmed at both the transcription and translation levels. Although MF is associated with a Th2 bias, Th1 cytokines IL-2 and IFN-gamma enhanced CTLA-4 expression, while IL-4 did not. These findings reveal an abnormal regulation of CTLA-4 expression in MF and show that PBMCs from patients with MF have properties that are divergent from those of normal T cells.