Translation of Data from Animal Models of Cancer to Immunotherapy of Breast Cancer and Chronic Lymphocytic Leukemia.

The field of clinical oncology has been revolutionized over the past decade with the introduction of many new immunotherapies the existence of which have depended to a large extent on experimentation with both in vitro analysis and the use of various animal models, including gene-modified mice. The discussion below will review my own laboratory's studies, along with those of others in the field, on cancer immunotherapy. Our own studies have predominantly dwelt on two models of malignancy, namely a solid tumor model (breast cancer) and lymphoma. The data from our own laboratory, and that of other scientists, highlights the novel information so obtained, and the evidence that application of such information has already had an impact on immunotherapy of human oncologic diseases.

High hypoxia status in pancreatic cancer is associated with multiple hallmarks of an immunosuppressive tumor microenvironment.

Introduction: Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, is a particularly lethal disease that is often diagnosed late and is refractory to most forms of treatment. Tumour hypoxia is a key hallmark of PDAC and is purported to contribute to multiple facets of disease progression such as treatment resistance, increased invasiveness, metabolic reprogramming, and immunosuppression. Methods: We used the Buffa gene signature as a hypoxia score to profile transcriptomics datasets from PDAC cases. We performed cell-type deconvolution and gene expression profiling approaches to compare the immunological phenotypes of cases with low and high hypoxia scores. We further supported our findings by qPCR analyses in PDAC cell lines cultured in hypoxic conditions. Results: First, we demonstrated that this hypoxia score is associated with increased tumour grade and reduced survival suggesting that this score is correlated to disease progression. Subsequently, we compared the immune phenotypes of cases with high versus low hypoxia score expression (HypoxiaHI vs. HypoxiaLOW) to show that high hypoxia is associated with reduced levels of T cells, NK cells and dendritic cells (DC), including the crucial cDC1 subset. Concomitantly, immune-related gene expression profiling revealed that compared to HypoxiaLOW tumours, mRNA levels for multiple immunosuppressive molecules were notably elevated in HypoxiaHI cases. Using a Random Forest machine learning approach for variable selection, we identified LGALS3 (Galectin-3) as the top gene associated with high hypoxia status and confirmed its expression in hypoxic PDAC cell lines. Discussion: In summary, we demonstrated novel associations between hypoxia and multiple immunosuppressive mediators in PDAC, highlighting avenues for improving PDAC immunotherapy by targeting these immune molecules in combination with hypoxia-targeted drugs.

Congenic hematopoietic stem cell transplantation promotes survival of heart allografts in murine models of acute and chronic rejection.

The ability to induce tolerance would be a major advance in the field of solid organ transplantation. Here, we investigated whether autologous (congenic) hematopoietic stem cell transplantation (HSCT) could promote tolerance to heart allografts in mice. In an acute rejection model, fully MHC-mismatched BALB/c hearts were heterotopically transplanted into C57BL/6 (CD45.2) mice. One week later, recipient mice were lethally irradiated and reconstituted with congenic B6 CD45.1 Lin-Sca1+ckit+ cells. Recipient mice received a 14-day course of rapamycin both to prevent rejection and to expand regulatory T cells (Tregs). Heart allografts in both untreated and rapamycin-treated recipients that did not undergo HSCT were rejected within 33 days (median survival time = 8 days for untreated recipients, median survival time = 32 days for rapamycin-treated recipients), whereas allografts in HSCT-treated recipients had a median survival time of 55 days (P < 0.001 vs. both untreated and rapamycin-treated recipients). Enhanced allograft survival following HSCT was associated with increased intragraft Foxp3+ Tregs, reduced intragraft B cells, and reduced serum donor-specific antibodies. In a chronic rejection model, Bm12 hearts were transplanted into C57BL/6 (CD45.2) mice, and congenic HSCT was performed two weeks following heart transplantation. HSCT led to enhanced survival of allografts (median survival time = 70 days vs. median survival time = 28 days in untreated recipients, P < 0.01). Increased allograft survival post-HSCT was associated with prevention of autoantibody development and absence of vasculopathy. These data support the concept that autologous HSCT can promote immune tolerance in the setting of allotransplantation. Further studies to optimize HSCT protocols should be performed before this procedure is adopted clinically.

Nature of Acquired Immune Responses, Epitope Specificity and Resultant Protection from SARS-CoV-2.

The primary global response to the SARS-CoV-2 pandemic has been to bring to the clinic as rapidly as possible a number of vaccines that are predicted to enhance immunity to this viral infection. While the rapidity with which these vaccines have been developed and tested (at least for short-term efficacy and safety) is commendable, it should be acknowledged that this has occurred despite the lack of research into, and understanding of, the immune elements important for natural host protection against the virus, making this endeavor a somewhat unique one in medical history. In contrast, as pointed out in the review below, there were already important past observations that suggested that respiratory infections at mucosal surfaces were susceptible to immune clearance by mechanisms not typical of infections caused by systemic (blood-borne) pathogens. Accordingly, it was likely to be important to understand the role for both innate and acquired immunity in response to viral infection, as well as the optimum acquired immune resistance mechanisms for viral clearance (B cell or antibody-mediated, versus T cell mediated). This information was needed both to guide vaccine development and to monitor its success. We have known that many pathogens enter into a quasi-symbiotic relationship with the host, with each undergoing sequential change in response to alterations the other makes to its presence. The subsequent evolution of viral variants which has caused such widespread concern over the last 3-6 months as host immunity develops was an entirely predictable response. What is still not known is whether there will be other unexpected side-effects of the deployment of novel vaccines in humans which have yet to be characterized, and, if so, how and if these can be avoided. We conclude by remarking that to ignore a substantial body of well-attested immunological research in favour of expediency is a poor way to proceed.

The efficient Lamarckian spread of life in the cosmos.

In this Chapter we discuss the various mechanisms that are available for the possible transfer of cosmic microbial living systems from one cosmic habitat to another. With the 100 or so habitable planets that are now known to exist in our galaxy alone transfers of cometary dust carrying life including fragments of icy planetoids/asteroids would be expected to occur on a routine basis. It is thus easy to view the galaxy as a single connected "biosphere" of which our planet Earth is a minor component. The Hoyle-Wickramasinghe Panspermia paradigm provides a cogent biological rationale for the actual widespread existence of Lamarckian modes of inheritance in terrestrial systems (which we review here). Thus the Panspermia paradigm provides the raison d'etre for Lamarckian Inheritance. Under a terrestrially confined neoDarwinian viewpoint such an association may have been thought spurious in the past. Our aim here is to outline the main evidence for rapid terrestrial-based Lamarckian-based evolutionary hypermutation processes dependent on reverse transcription-coupled mechanisms among others. Such rapid adaptation mechanisms would be consistent with the effective cosmic spread of living systems. For example, a viable, or cryo-preserved, living system traveling through space in a protective matrix will of necessity need to adapt rapidly and proliferate on landing in a new cosmic niche. Lamarckian mechanisms thus come to the fore and supersede the slow (blind and random) genetic processes expected under neoDarwinian Earth centred theories.

Origin of new emergent Coronavirus and Candida fungal diseases-Terrestrial or cosmic?

The origins and global spread of two recent, yet quite different, pandemic diseases is discussed and reviewed in depth: Candida auris, a eukaryotic fungal disease, and COVID-19 (SARS-CoV-2), a positive strand RNA viral respiratory disease. Both these diseases display highly distinctive patterns of sudden emergence and global spread, which are not easy to understand by conventional epidemiological analysis based on simple infection-driven human- to-human spread of an infectious disease (assumed to jump suddenly and thus genetically, from an animal reservoir). Both these enigmatic diseases make sense however under a Panspermia in-fall model and the evidence consistent with such a model is critically reviewed.

CD200 mimetic aptamer PEG-M49 markedly increases the therapeutic effects of pegylated liposomal doxorubicin in a mouse model of metastatic breast carcinoma: an effect independent of CD200 receptor 1.

We previously reported that CD200 overexpression in the host decreases progression and metastasis of the highly aggressive metastatic 4THM breast carcinoma. We have explored a possible synergistic interaction between the CD200 mimetic PEG-M49 and pegylated liposomal doxorubicin (Peg-Dox) in wild-type CD200 knockout (CD200-/-) and CD200 Receptor 1 knockout (CD200R1-/-) mice for the first time. A 4THM breast carcinoma model and three groups of BALB/c mice (wild type, CD200-/- and CD200R1-/-) were used. Five days after injection of tumor cells, mice were injected with Peg-Dox (ip, once a week) and PEG-M49 or a control aptamer (iv, every 3 days). Necropsies were performed either 12 (mid-point) or 24 (endpoint) days after injection and the extent of tumor growth, visceral metastasis and changes in the tumor-directed immune response were evaluated. PEG-M49 and Peg-Dox co-treatment induced complete tumor regression and loss of macroscopic lung metastasis in four out of seven WT mice. This synergistic anti-tumoral effect is thought to be due to Peg-M49-induced inhibition of Gr1 + CD11b + cells and Peg-Dox-induced increases in tumor-infiltrating CD8 + and CD8CD4 double-positive cells. Similar changes were observed in CD200R1-/- mice indicating that the primary effects of Peg-M49 are mediated by non-CD200R1 receptors. We also demonstrated for the first time that tumor growth, metastasis, and tumor infiltrating GR1 + CD11b + cells were markedly increased in CD200R1-/- mice, indicating an anti-inflammatory and protective role of CD200. CD200 mimetics might be a safe and effective immunomodulatory treatment in conjunction with classical chemotherapeutics for therapy of aggressive metastatic breast carcinoma.

Lamarck and Panspermia - On the Efficient Spread of Living Systems Throughout the Cosmos.

We review the main lines of evidence (molecular, cellular and whole organism) published since the 1970s demonstrating Lamarckian Inheritance in animals, plants and microorganisms viz. the transgenerational inheritance of environmentally-induced acquired characteristics. The studies in animals demonstrate the genetic permeability of the soma-germline Weismann Barrier. The widespread nature of environmentally-directed inheritance phenomena reviewed here contradicts a key pillar of neo-Darwinism which affirms the rigidity of the Weismann Barrier. These developments suggest that neo-Darwinian evolutionary theory is in need of significant revision. We argue that Lamarckian inheritance strategies involving environmentally-induced rapid directional genetic adaptations make biological sense in the context of cosmic Panspermia allowing the efficient spread of living systems and genetic innovation throughout the Universe. The Hoyle-Wickramasinghe Panspermia paradigm also developed since the 1970s, unlike strictly geocentric neo-Darwinism provides a cogent biological rationale for the actual widespread existence of Lamarckian modes of inheritance - it provides its raison d'être. Under a terrestrially confined neo-Darwinian viewpoint such an association may have been thought spurious in the past. Our aim is to outline the conceptual links between rapid Lamarckian-based evolutionary hypermutation processes dependent on reverse transcription-coupled mechanisms among others and the effective cosmic spread of living systems. For example, a viable, or cryo-preserved, living system travelling through space in a protective matrix will need of necessity to rapidly adapt and proliferate on landing in a new cosmic niche. Lamarckian mechanisms thus come to the fore and supersede the slow (blind and random) genetic processes expected under a traditional neo-Darwinian evolutionary paradigm.

A Synthetic Cross-Species CD200R1 Agonist Suppresses Inflammatory Immune Responses In Vivo.

Functional aptamers displaying agonistic or antagonistic properties are showing great promise as modulators of immune responses. Here, we report the development of a polyethylene glycol-modified (PEGylated) DNA aptamer as a cross-species (murine and human) CD200R1 agonist that modulates inflammatory responses in vivo. Specifically, DNA aptamers were discovered by performing independent SELEX searches on recombinant murine and human CD200R1. Aptamer motifs identified by next generation sequencing (NGS) were subsequently compared, leading to the discovery of motifs common to both targets. The CD200R1 DNA aptamer CCS13 displayed the highest agonistic activity toward CD200R1 in terms of suppressing the induction of cytotoxic T-lymphocytes (CTLs) in both human and murine allogeneic-mixed lymphocyte cultures (allo-MLCs). A 20-kDa polyethylene glycol (PEG) chain was covalently attached to the 5' end of this aptamer, and the resulting conjugate was shown to block inflammatory responses in murine models of skin graft rejection and house-dust-mite-induced allergic airway inflammation. Importantly, this agonistic aptamer does not suppress CTL induction in 5-day allo-MLCs with responder cells derived from CD200R1-/- mice, indicating that its mode of action is directly linked to CD200R1 activation. This study suggests that one can derive agonistic DNA aptamers that can be verified as immuno-modulators in murine models with outcomes potentially translatable to the treatment of human conditions.

Cell membrane-bound CD200 signals both via an extracellular domain and following nuclear translocation of a cytoplasmic fragment.

In previous studies we had reported that the immunosuppressive cell membrane bound molecule CD200 is released from the cell following cleavage by matrix metalloproteases, with the released soluble CD200 acting as an immunosuppressant following binding to, and signaling through, its cognate receptor CD200R expressed on target cells. We now show that although the intracellular cytoplasmic tail (CD200C-tail) of CD200 has no consensus sites for adapter molecules which might signal the CD200+ cell directly, cleavage of the CD200C-tail from the membrane region of CD200 by a consensus γ-secretase, leads to nuclear translocation and DNA binding (identified by chromatin immunoprecipitation followed by sequencing, Chip-sequencing) of the CD200C-tail. Subsequently there occurs an altered expression of a limited number of genes, many of which are transcription factors (TFs) known to be associated with regulation of cell proliferation. Altered expression of these TFs was also prominent following transfection of CD200+ B cell lines and fresh patient CLL cells with a vector construct containing the CD200C-tail. Artificial transfection of non CD200+ Hek293 cells with this CD200C-tail construct resulted in altered expression of most of these same genes. Introduction of a siRNA for one of these TFs, POTEA, reversed CD200C-tail regulation of altered cell proliferation.

Cause of Cambrian Explosion - Terrestrial or Cosmic?

We review the salient evidence consistent with or predicted by the Hoyle-Wickramasinghe (H-W) thesis of Cometary (Cosmic) Biology. Much of this physical and biological evidence is multifactorial. One particular focus are the recent studies which date the emergence of the complex retroviruses of vertebrate lines at or just before the Cambrian Explosion of ∼500 Ma. Such viruses are known to be plausibly associated with major evolutionary genomic processes. We believe this coincidence is not fortuitous but is consistent with a key prediction of H-W theory whereby major extinction-diversification evolutionary boundaries coincide with virus-bearing cometary-bolide bombardment events. A second focus is the remarkable evolution of intelligent complexity (Cephalopods) culminating in the emergence of the Octopus. A third focus concerns the micro-organism fossil evidence contained within meteorites as well as the detection in the upper atmosphere of apparent incoming life-bearing particles from space. In our view the totality of the multifactorial data and critical analyses assembled by Fred Hoyle, Chandra Wickramasinghe and their many colleagues since the 1960s leads to a very plausible conclusion - life may have been seeded here on Earth by life-bearing comets as soon as conditions on Earth allowed it to flourish (about or just before 4.1 Billion years ago); and living organisms such as space-resistant and space-hardy bacteria, viruses, more complex eukaryotic cells, fertilised ova and seeds have been continuously delivered ever since to Earth so being one important driver of further terrestrial evolution which has resulted in considerable genetic diversity and which has led to the emergence of mankind.

An autologous tumor vaccine for CLL.

Chronic Lymphocytic Leukemia B cells (CLL) are malignant cells which retain at least some functions of normal B cells. Paramount amongst the latter is that when such cells are appropriately stimulated, they are able to present antigens, including any potential tumor antigens, making them excellent choices as a candidate tumor vaccine. We show that following stimulation of CLL cells with Phorbol myristic acetate, IL-2, the TLR7 agonist imiquimod (P2I) and ionomycin (P2Iio), markedly increased expression of CD54 and CD83 was seen, indicative of B cell activation and a transition to antigen-presenting cells. However, this occurred in the context of augmented expression of the known immunoregulatory molecule, CD200. Accordingly we explored the effect of stimulation of CLL cells with P2Iio, followed by coating of cells with a non-depleting anti-CD200mAb, on the ability of those cells to immunize PBL in vitro to become cytotoxic to CLL cells, or to protect NOD-SCIDγcnull (NSG) mice from subsequent CLL tumor challenge. Our data indicate that this protocol is effective in inducing CD8+ CTL able to lyse CLL cells in vitro, and decrease tumor burden in vivo in spleen and marrow of mice injected with CLL cells. Pre-treatment of mice with a CD8 depleting antibody before vaccination with P2Iio/anti-CD200 coated cells abolished any protection seen. These data suggest a potential role for blockade of CD200 expression on CLL cells as a component of a tumor vaccination strategy.

Characterization of an in vitro model system to explore control of tumor invasion of EMT6 and 4THM breast tumors by CD200:CD200R interactions.

BACKGROUND AND PURPOSE: In BALB/c mice with transplantable breast tumors, we showed that CD200R1KO mice were cured of local and metastatic growth of EMT6 cells following surgical resection of localized tumor and immunization with irradiated cells along with CpG as adjuvant. On the other hand, wild-type (WT) animals treated in the same fashion develop pulmonary and liver metastases within 20 days of surgery. To develop an in vitro system which would mimic the in vivo model and allow exploration of factors controlling tumor invasion as a precursor to in vivo metastasis, we have developed and characterized a two-phase culture system. METHODS: Bone marrow mesenchymal stromal cells (BMMSCs) from WT, CD200KO or CD200R1KO mice were admixed with T lymphocytes from tumor-immunized mice and cultured in collagen gels. Tumor cells were subsequently seeded in fresh medium above this gel 1d later. We then investigated the regulation of tumor invasion from the liquid to the gel layer. Tumor cells were measured in the gel layer following collagenase digestion and cultured at limiting dilution-an aliquot of the digest was also analyzed for cytokine levels in ELISA. RESULTS: BMMSCs from WT, CD200KO and CD200R1KO mice all augmented seeding/growth of EMT6 and 4THM tumor cells into the collagen matrix. Inclusion of IL-6 and IL-17 in the gel matrix was associated with increased invasion of tumor cells into this layer. Inclusion of DLN cells from EMT6 immune or 4THM immune mice further modified tumor invasion, with increased tumor numbers seen using stromal elements from CD200 and CD200R1KO mice and DLN from 4THM immune, while CD200R1KO-derived DLN of EMT6 immune mice attenuated tumor invasion, despite inclusion of IL-6/IL-17 in the gel layer. CONCLUSION: Multiple factors can regulate tumor invasion, including micro-environmental stromal elements, IL-6/IL-17, and signals from tumor-derived DLN cells.

Importance of B cells to development of regulatory T cells and prolongation of tissue allograft survival in recipients receiving autologous bone marrow transplantation.

We previously showed that congenic bone marrow transplantation (BMTx) post myeloablation augmented tissue allograft survival in association with increased regulatory T (Treg) cells of both host and bone marrow donor origin. Regulatory B (Breg) cells can also modulate T-cell immunity and B cells may be implicated in the development of Treg cells. Accordingly, we explored the effect of B-cell depletion in vivo on augmented graft survival post BMTx. C57BL/6 mice received BALB/c skin allografts followed 7 days later by myeloablation using cyclophosphamide and busulphan. Mice then received T-cell-depleted bone marrow from CD45.1 congenic donors, and ongoing immunosuppression with rapamycin (to day 28 after BMTx). Control mice received cyclophosphamide and busulphan followed by rapamycin, but not congenic bone marrow. At different times post BMTx, mice received B-cell-depleting antibody treatment, and the effect on both skin graft survival, and induction of Treg cells was assessed. BMTx resulted in significantly prolonged skin graft survival versus control mice, in association with attenuated donor-specific alloreactivity relative to controls, increased splenic Treg cells and significantly diminished anti-donor IgG. In mice receiving infusion of B-depleting antibodies for 12 days from day 15 post BMTx, both graft survival and Treg cell activity were diminished, particularly for functional Treg cells of donor origin. Adoptive transfer of Breg cells from mice harvested at 15 days post BMTx prolonged survival in naive transplanted mice and increased Treg cell levels. Thus, autologous BMTx augmentation of graft survival is dependent in part upon a population of Breg cells that can modulate the function of donor-derived Treg cells.

Targeting the IL-17/IL-6 axis can alter growth of Chronic Lymphocytic Leukemia in vivo/in vitro.

The tumor microenvironment (TME) is critical to the longevity of tumor B cells in chronic lymphocytic leukemia (CLL). Bone marrow mesenchymal stem cells (BMMSCs) and the cytokines they produce including IL-6 are important components of the TME in CLL. We found BMMSCs supported the survival of CLL cells in vitro through an IL-6 dependent mechanism. IL-17 which induces IL-6 generation in a variety of cells increased production of IL-6 both in CLL cells and BMMSCs in vitro. In a xenograft CLL mouse model, BMMSCs and the culture supernatant of BMMSCs increased engraftment of CLL cells through an IL-6 mediated mechanism with human recombinant IL-6 showing similar effects in vivo. Human recombinant IL-17 treatment also increased CLL engraftment in mice through an IL-6 mediated mechanism. Plasma of CLL patients showed elevated levels of both IL-6 and IL-17 by ELISA compared with healthy controls, with levels of IL-6 linearly correlated with IL-17 levels. CLL patients requiring fludarabine based chemotherapy expressed higher levels of IL-6 and IL-17, while CLL patients with the lowest levels of IgA/IgM had higher levels of IL-6, but not IL-17. These data imply an important role for the IL-17/IL-6 axis in CLL which could be therapeutic targets.