Response to COVID-19 vaccination in patients on cancer therapy: Analysis in a SARS-CoV-2-naïve population.

BACKGROUND: Cancer patients have increased morbidity and mortality from COVID-19, but may respond poorly to vaccination. The Evaluation of COVID-19 Vaccination Efficacy and Rare Events in Solid Tumors (EVEREST) study, comparing seropositivity between cancer patients and healthy controls in a low SARS-CoV-2 community-transmission setting, allows determination of vaccine response with minimal interference from infection. METHODS: Solid tumor patients from The Canberra Hospital, Canberra, Australia, and healthy controls who received COVID-19 vaccination between March 2021 and January 2022 were included. Blood samples were collected at baseline, pre-second vaccine dose and at 1, 3 (primary endpoint), and 6 months post-second dose. SARS-CoV-2 anti-spike-RBD (S-RBD) and anti-nucleocapsid IgG antibodies were measured. RESULTS: Ninety-six solid tumor patients and 20 healthy controls were enrolled, with median age 62 years, and 60% were female. Participants received either AZD1222 (65%) or BNT162b2 (35%) COVID-19 vaccines. Seropositivity 3 months post vaccination was 87% (76/87) in patients and 100% (20/20) in controls (p = .12). Seropositivity was observed in 84% of patients on chemotherapy, 80% on immunotherapy, and 96% on targeted therapy (differences not satistically significant). Seropositivity in cancer patients increased from 40% (6/15) after first dose, to 95% (35/37) 1 month after second dose, then dropped to 87% (76/87) 3 months after second dose. CONCLUSION: Most patients and all controls became seropositive after two vaccine doses. Antibody concentrations and seropositivity showed a decrease between 1 and 3 months post vaccination, highlighting need for booster vaccinations. SARS-CoV-2 infection amplifies S-RBD antibody responses; however, cannot be adequately identified using nucleocapsid serology. This underlines the value of our COVID-naïve population in studying vaccine immunogenicity.

Early T Cell Infiltration Correlates with Anti-CTLA4 Treatment Response in Murine Cancer Models.

Immune checkpoint inhibitor (ICI) Abs are a revolutionary class of cancer treatment, but only ∼30% of patients receive a lasting benefit from therapy. Preclinical studies using animals from the same genetic backgrounds, challenged with the same cancer models, also show nonuniform responses. Most mouse studies that have evaluated tumor-infiltrating leukocytes after ICI therapy cannot directly correlate their findings with treatment outcomes, because terminal methods were used to acquire immune infiltrate data. In the present study, we used fine-needle aspiration (a nonterminal sampling method) to collect multiple aspirates over several days from s.c. implanted P815, CT26, and 4T1 mouse cancer models treated with ICI Abs. These aspirates were then analyzed with flow cytometry to directly correlate tumor-infiltrating leukocyte populations with treatment success. We found that the P815 and CT26 models respond well to anti-CTLA4 therapies. Among P815-challenged animals, mice that regressed following anti-CTLA4 treatment showed significant increases in CD8+ T cells on days 3, 5, and 7 and in CD4+ T cells on days 5 and 7 and a decrease in macrophages and monocytes on days 3, 5, and 7 after treatment. Similar results were obtained in the CT26 model on day 11 posttreatment. Our study is the first, to our knowledge, to directly correlate early tumor infiltration of T cells with anti-CTLA4 treatment success, thus providing a mechanistic clue toward understanding why alloidentical mice challenged with identical tumors do not respond uniformly to ICI therapies.

Gut microbiome-depleting antibiotic regimens are not tolerated by all mouse strains: learn from (our) bitter experience.

Why the gut microbiome is critical for the success of checkpoint inhibitor cancer therapy is a question that remains unanswered, but progress has slowed. We argue that this lack of advancement is due to an unappreciated biological detail. Here, we show that the antibiotic cocktail used in seminal publications-all of which have used the C57BL/6 mouse strain-are bitter and not tolerated by other common mouse strains (ie, BALB/c and DBA/2). We write to alert readers of this important biological limitation that must be considered when planning cancer experiments investigating the gut microbiota, to prevent the unnecessary dehydration of experimental animals, and to save our colleagues valuable experimental time and resources.

Simple and effective bacterial-based intratumoral cancer immunotherapy.

BACKGROUND: We describe intratumoral injection of a slow-release emulsion of killed mycobacteria (complete Freund's adjuvant (CFA)) in three preclinical species and in human cancer patients. METHODS: Efficacy and safety were tested in mammary tumors in mice, in mastocytomas in mice and dogs, and in equine melanomas. In mice, survival, tumor growth, and tumor infiltration by six immune cell subsets (by flow cytometry) were investigated and analyzed using Cox proportional hazards, a random slopes model, and a full factorial model, respectively. Tumor growth and histology were investigated in dogs and horses, as well as survival and tumor immunohistochemistry in dogs. Tumor biopsies were taken from human cancer patients on day 5 (all patients) and day 28 (some patients) of treatment and analyzed by histology. CT scans are provided from one patient. RESULTS: Significantly extended survival was observed in mouse P815 and 4T1 tumor models. Complete tumor regressions were observed in all three non-human species (6/186 (3%) of mouse mastocytomas; 3/14 (21%) of canine mastocytomas and 2/11 (18%) of equine melanomas). Evidence of systemic immune responses (regression of non-injected metastases) was also observed. Analysis of immune cells infiltrating mastocytoma tumors in mice showed that early neutrophil infiltration was predictive of treatment benefit. Analysis of the site of mastocytoma regression in dogs weeks or months after treatment demonstrated increased B and T cell infiltrates. Thus, activation of the innate immune system alone may be sufficient for regression of some injected tumors, followed by activation of the acquired immune system which can mediate regression of non-injected metastases. Finally, we report on the use of CFA in 12 human cancer patients. Treatment was well tolerated. CT scans showing tumor regression in a patient with late-stage renal cancer are provided. CONCLUSION: Our data demonstrate that intratumoral injection of CFA has major antitumor effects in a proportion of treated animals and is safe for use in human cancer patients. Further trials in human cancer patients are therefore warranted. Our novel treatment provides a simple and inexpensive cancer immunotherapy, immediately applicable to a wide range of solid tumors, and is suitable to patients in developing countries and advanced care settings.

How does the gut microbiome influence immune checkpoint blockade therapy?

Immune checkpoint blockade (ICB) therapies are revolutionary cancer treatments; however, they only benefit about a third of patients. Therefore, extensive research is underway to find methods to improve their therapeutic efficacy. One avenue of study that has recently emerged is to consider the role the gut microbiome plays in therapeutic success. Several high-impact studies have repeatedly shown that the presence, composition and level of diversity of the gut flora directly impact cancer treatment outcome in both mice and patients. These studies have also highlighted the danger of using antibiotics shortly before or during cancer treatments. However, there are still several questions that need to be answered, including which bacteria promote the greatest benefit, the mechanisms by which they act and how we can use this information to influence treatment outcome. In this review, we explain how the gut microbiome was realized to be of such importance and propose hypotheses for why gut flora have such a critical impact on ICB therapeutic success. We also describe a hypothetical mechanism involving bacterial translocation out of the gut and into the tumor, whereby the bacteria act in an adjuvant capacity to facilitate an antitumor response. By highlighting key papers in the field, we hope to hasten research on the subject so as to find a means to improve the therapeutic efficacy of these ground-breaking cancer treatments.

Repeated fine-needle aspiration of solid tumours in mice allows the identification of multiple infiltrating immune cell types.

This paper describes a novel method for following the changes in mouse tumour-infiltrating immune cell populations by repeated sampling of tumours by fine needle aspiration (FNA), followed by flow cytometry. Using this technique we were able to collect samples from P815 mouse mastocytomas, and identify and enumerate six tumour-infiltrating immune cell types at multiple time points for each mouse. We demonstrate good agreement between cell percentages obtained from FNA samples and matched whole tumour digests (WTDs). We also demonstrate that neither survival nor the incidence of liver metastasis is adversely affected by the procedure. Our method has a clear advantage over the common practice of sacrificing mice and collecting tissue at pre-determined time points, as the technique allows 1) repeated sampling of each mouse over time, thus many fewer mice are required, and 2) the correlation of survival data with tumour-infiltrating immune cell types at different time points. This potentially allows immune cell types associated with increased or decreased survival to be identified. Therefore, our technique should greatly facilitate the characterisation of anti-tumour immunity induced in response to cancer therapy in small animal models.

A novel splicing outcome reveals more than 2000 new mammalian protein isoforms.

MOTIVATION: We have recently characterized an instance of alternative splicing that differs from the canonical gene transcript by deletion of a length of sequence not divisible by three, but where translation can be rescued by an alternative start codon. This results in a predicted protein in which the amino terminus differs markedly in sequence from the known protein product(s), as it is translated from an alternative reading frame. Automated pipelines have annotated thousands of splice variants but have overlooked these protein isoforms, leading to them being underrepresented in current databases. RESULTS: Here we describe 1849 human and 733 mouse transcripts that can be transcribed from an alternate ATG. Of these, >80% have not been annotated previously. Those conserved between human and mouse genomes (and hence under likely evolutionary selection) are identified. We provide mass spectroscopy evidence for translation of selected transcripts. Of the described splice variants, only one has previously been studied in detail and converted the encoded protein from an activator of cell-function to a suppressor, demonstrating that these splice variants can result in profound functional change. We investigate the potential functional effects of this splicing using a variety of bioinformatic tools. The 2582 variants we describe are involved in a wide variety of biological processes, and therefore open many new avenues of research.

Splice variants of the condensin II gene Ncaph2 include alternative reading frame translations of exon 1.

Condensins I and II are five-protein complexes that are important for the condensation of chromatin. They are essential for mitosis and important for regulating gene expression during interphase. Here, we investigated the transcription and translation of the mouse Ncaph2 gene, which encodes a subunit of condensin II. We identified three splice variants within the first exon, a NAGNAG splice variant at the beginning of exon 16 and alternative 3'-UTRs. In total, Ncaph2 is potentially capable of generating 12 unique mRNA transcripts and six unique proteins. We confirm that Ncaph2 can generate three different N-termini, all encoded by exon 1, one of which is translated from an alternative reading frame. This alternative reading frame splice variant appears to be a novel outcome of splicing. If this is applicable to other genes, it would account for a previously unappreciated level of eukaryotic protein diversity.

A proposal for a simple and inexpensive therapeutic cancer vaccine.

In this essay, I propose a new method of treating tumours, using an old and inexpensive preparation, that I contend would be of considerable benefit to patients and their cancer management. My rationale for this treatment initially arose from recent advances in the understanding of dendritic cell function. (Dendritic cells are key cells of the immune system that are able to either turn on or turn off T-cell responses.) Evidence to support this approach is found in 100-year-old studies on the immunotherapy of cancer. Also, I draw on some remarkable, but little-known studies from the 1960s-1990s, demonstrating that the preparation has already been trialled in humans (although not intratumourally, as I propose), and is considered sufficiently safe to proceed with clinical trials in cancer volunteers.

Analysis of A47, an immunoprevalent protein of vaccinia virus, leads to a reevaluation of the total antiviral CD8+ T cell response.

Vaccinia virus (VACV) is the prototypic orthopoxvirus and was the live vaccine used to eradicate smallpox. In addition, VACV is a possible vector for recombinant vaccines. Despite these reasons for study, the roles of many VACV genes are unknown, and some fundamental aspects, such as the total size of immune responses, remain poorly characterized. VACV gene A47L is of interest because it is highly transcribed, has no sequence similarity to any nonpoxvirus gene, and contains a larger-than-expected number of CD8(+) T cell epitopes. Here it is shown that A47L is not required for growth in vitro and does not contribute to virulence in mice. However, we confirmed that this one protein primes CD8(+) T cells to three different epitopes in C57BL/6 mice. In the process, one of these epitopes was redefined and shown to be the most dominant in A47 and one of the more highly ranked in VACV as a whole. The relatively high immunogenicity of this epitope led to a reevaluation of the total CD8(+) T cell response to VACV. By the use of two methods, the true size of the response was found to be around double previous estimates and at its peak is on the order of 60% of all CD8(+) T cells. We speculate that more CD8(+) T cell epitopes remain to be mapped for VACV and that underestimation of responses is unlikely to be unique to VACV, so there would be merit in revisiting this issue for other viruses.

Mouse strains with point mutations in TAP1 and TAP2.

We report two new mouse strains: Jasmine (C57BL/6J/Apb-Tap2jas/Apb), with a point mutation in the transporter associated with antigen processing (TAP)2 ; and Rose, (C57BL/6J/Apb-Tap1rose/Apb), with a point mutation in TAP1. These strains were detected as the result of ethyl nitroso urea (ENU) screens for recessive point mutations affecting the immune system. As expected in cases of defective TAP expression, the mice have very low major histocompatibility complex (MHC)-I cell-surface expression, and few CD8(+) T cells. The Rose strain has an A to T substitution in exon 10 of TAP1, resulting in an asparagine to valine substitution at position 643. Jasmine has an A to C transversion in exon 5 of TAP2, resulting in a threonine to proline substitution at position 293 of the protein. The mutation does not affect mRNA levels, but results in a very severe reduction in TAP2 protein. TAP1 protein levels are also decreased in Jasmine mice, demonstrating a new role for mouse TAP2 in stabilizing TAP1 protein expression. Jasmine is the first strain available with defective TAP2. The two mouse strains provide additional animal models for the human condition Bare Lymphocyte syndrome type 1, and identify new residues important for TAP function.

Defective T-cell function leading to reduced antibody production in a kleisin-beta mutant mouse.

The recently described nessy (Ncaph2nes/nes) mutant mouse strain has a defect in T-cell development caused by a mutation in the ubiquitous kleisin-beta (also known as Ncaph2). Kleisin-beta is a subunit of the condensin II complex involved in chromosome condensation during mitosis. The nessy phenotype is characterized by CD44hi CD8+ peripheral T cells, 10-20% of normal thymocyte numbers and 2.5-fold fewer alphabeta T cells in the spleen compared with wild-type mice. In this study we examined the effect of the nessy mutation in kleisin-beta on the immune response by challenging mice with an attenuated strain of Salmonella. Results showed that nessy mice control bacterial load as effectively as wild-type mice but exhibit a reduced antibody titre. Further experiments revealed that while the T-dependent antibody response was diminished in nessy mice the T-independent response was normal, suggesting that the defect was the result of T-cell function and not B-cell function. In vitro activation assays showed that nessy T cells have a lower capacity to up-regulate the early activation marker CD69 than wild-type T cells. Upon transfer into RAG-/- mice, nessy and wild-type CD4 T cells showed equivalent homeostatic proliferation, while nessy CD8 T cells proliferated more than their wild-type counterparts. When cultured with anti-T-cell receptor beta or concanavalin A, nessy T cells were found to die faster than wild-type T cells. These data indicate that kleisin-beta is required for a normal immune response, and represent the first demonstration of a role for kleisin-beta in T-cell function.

A mutation in a chromosome condensin II subunit, kleisin beta, specifically disrupts T cell development.

Condensins are ubiquitously expressed multiprotein complexes that are important for chromosome condensation and epigenetic regulation of gene transcription, but whose specific roles in vertebrates are poorly understood. We describe a mouse strain, nessy, isolated during an ethylnitrosourea screen for recessive immunological mutations. The nessy mouse has a defect in T lymphocyte development that decreases circulating T cell numbers, increases their expression of the activation/memory marker CD44, and dramatically decreases the numbers of CD4(+)CD8(+) thymocytes and their immediate DN4 precursors. A missense mutation in an unusual alternatively spliced first exon of the kleisin beta gene, a member of the condensin II complex, was shown to be responsible and act in a T cell-autonomous manner. Despite the ubiquitous expression and role of condensins, kleisin beta(nes/nes) mice were viable, fertile, and showed no defects even in the parallel pathway of B cell lymphocyte differentiation. These data define a unique lineage-specific requirement for kleisin beta in mammalian T cell differentiation.

Impairment of organ-specific T cell negative selection by diabetes susceptibility genes: genomic analysis by mRNA profiling.

BACKGROUND: T cells in the thymus undergo opposing positive and negative selection processes so that the only T cells entering circulation are those bearing a T cell receptor (TCR) with a low affinity for self. The mechanism differentiating negative from positive selection is poorly understood, despite the fact that inherited defects in negative selection underlie organ-specific autoimmune disease in AIRE-deficient people and the non-obese diabetic (NOD) mouse strain RESULTS: Here we use homogeneous populations of T cells undergoing either positive or negative selection in vivo together with genome-wide transcription profiling on microarrays to identify the gene expression differences underlying negative selection to an Aire-dependent organ-specific antigen, including the upregulation of a genomic cluster in the cytogenetic band 2F. Analysis of defective negative selection in the autoimmune-prone NOD strain demonstrates a global impairment in the induction of the negative selection response gene set, but little difference in positive selection response genes. Combining expression differences with genetic linkage data, we identify differentially expressed candidate genes, including Bim, Bnip3, Smox, Pdrg1, Id1, Pdcd1, Ly6c, Pdia3, Trim30 and Trim12. CONCLUSION: The data provide a molecular map of the negative selection response in vivo and, by analysis of deviations from this pathway in the autoimmune susceptible NOD strain, suggest that susceptibility arises from small expression differences in genes acting at multiple points in the pathway between the TCR and cell death.

Generalized resistance to thymic deletion in the NOD mouse; a polygenic trait characterized by defective induction of Bim.

The cause of common polygenic autoimmune diseases is not understood because of genetic and cellular complexity. Here, we pinpoint the action of a subset of autoimmune susceptibility loci in the NOD mouse strain linked to D1mit181, D2mit490, D7mit101, and D15mit229, which cause a generalized resistance to thymic deletion in vivo that applies equally to Aire-induced organ-specific gene products in the thymic medulla and to systemic antigens expressed at high levels throughout the thymus and affects CD4(+), CD4(+)8(+), and CD4(+)25(+) thymocytes. Resistance to thymic deletion does not reflect a general deficit in TCR signaling to calcineurin- or ERK-induced genes, imbalance in constitutive regulators of apoptosis, nor excessive signaling to prosurvival genes but is distinguished by failure to induce the proapoptotic gene and protein, Bim, during in vivo encounter with high-avidity autoantigen. These findings establish defects in thymic deletion and Bim induction as a key mechanism in the pathogenesis of autoimmunity.