Patent Application
20220053740
The present invention relates generally to the field of immunology. More particularly, it concerns an animal model for the autoimmune disease etiologies that arise as a result of immune checkpoint blockade therapies as well as methods of using the animal model to screen for agents that mitigate said disease etiologies.
T cell activation is an exquisitely regulated biological process that enables the generation of rapid and highly sensitive responses to foreign antigens while maintaining the ability to distinguish self from non-self and prevent autoimmunity. A key concept underlying this remarkable process is that multiple distinct signals are required to fully activate, or prime, naïve T cells. These cues include cognate antigen recognition by the T-cell receptor (TCR) (Signal 1) and CD28 positive co-stimulation (Signal 2). Because CD28 positive co-stimulation is provided by professional antigen presenting cells, this enforces a cell extrinsic requirement for robust T cell activation. The next key step is negative co-stimulation, which is a feedback regulatory mechanism that attenuates T cell activation through inhibition of Signals 1 and 2. CTLA4 and PD-1 are principal negative costimulatory molecules that attenuate T cell activation through distinct molecular mechanisms. While CTLA4 attenuates T cell activation via competitive inhibition of CD28 positive co-stimulation, PD-1 primarily acts to inhibit proximal T-cell receptor (TCR) signaling via the phosphatase SHP2 (Chemnitz et al., 2004; Krummel & Allison, 1996; Parry et al., 2005; Walunas et al., 1996). Recent evidence suggests that PD-1 also leads to inhibition of CD28 positive costimulation (Hui et al., 2017) and relatedly, that CD28 signaling is required for effective responses to PD-1 blockade (Kamphorst et al., 2017). This suggests that attenuation of CD28 may be a shared mechanism of PD-1 and CTLA4 mediated T cell regulation.
T cell activation is generally thought to be governed by a threshold model, in which TCR and costimulatory signals must meet a minimum level to trigger activation. It is unknown whether PD-1 and CTLA4 negative co-stimulation lead to convergent functional regulation or alternatively, whether they exert distinct regulatory pressures to define the activation threshold. Relatedly, the relative functional contribution of the specific mechanisms of CTLA4 and PD-1 to T cell attenuation remains unclear. It is possible that these distinct mechanisms converge at the molecular, cellular, and/or tissue level. For example, at the molecular level, CTLA4 and PD-1 may co-regulate T cell signaling in a cell intrinsic manner through inhibition of CD28. At the cellular level, CTLA4 and PD-1 attenuate T cells with distinct kinetics with respect to activation and it is unclear whether and how such temporally separated regulation is integrated. Thus, a critical open fundamental question is whether the distinct regulatory mechanisms of CTLA4 and PD-1 negative co-stimulation functionally interact.
Anti-CTLA4 and anti-PD-1 therapies are effective in multiple tumor types advanced melanoma and renal cell carcinoma. However, immune checkpoint blockade therapy can induce serious immune-related adverse events as well as bona fide autoimmunity, such as myocarditis and type I diabetes in rare instances. There are currently no animal models that faithfully recapitulate the adverse events associated with checkpoint blockade therapy. Treatment of mice with checkpoint blockade antibodies (i.e. anti-CTLA4, anti-PD-1) does not lead to significant pathologies and does not faithfully recapitulate the range, severity, and type of immune related adverse events seen in human patients. In particular, these models do not recapitulate the rare autoimmune diseases that are associated with checkpoint blockade. As such, animal models that recapitulate the adverse events associated with checkpoint blockade therapy are needed.
Provided herein is an animal model that recapitulates autoimmunity induced by checkpoint blockade in human patients. In one embodiment, a mouse is provided whose genome comprises: (i) a heterozygous loss-of-function of a Ctla4 gene and (ii) a homozygous loss-of-function of a Pdcd1 gene. In one aspect, the mouse has a C57BL/6J genetic background. In some aspects, the mouse is a female mouse. In some aspects, the mouse is a male mouse.
In some aspects, the heterozygous loss-of-function allele of a Ctla4 gene is further defined as a heterozygous insertion of a neomycin resistance cassette into exon 3 of the Ctla4 gene. In some aspects, the homozygous loss-of-function allele of the Pdcd1 gene is further defined as a homozygous deletion of exons 2 and 3 of the Pdcd1 gene. In one aspect, the mouse is a Ctla4tm1AllPdcd1tm1.1Shr mouse.
In some aspects, the mouse suffers from autoimmunity. In certain aspects, the autoimmunity is cardiac autoimmunity or pancreatic autoimmunity. In one aspect, the cardiac autoimmunity is myocarditis. In certain aspects, the myocarditis is fulminant myocarditis. In one aspect, the pancreatic autoimmunity is insulin-dependent diabetes mellitus or lymphocytic pancreatitis. In some aspects, the pancreatic autoimmunity results in pancreatic exocrine destruction or pancreatic islet destruction. In some aspects, the autoimmunity is lymphocytic myocarditis, endarteritis, pancreatic exocrine destruction, pulmonary vasculitis, adipose tissue atrophy (both white and brown), hepatic inflammation, atrophy of female reproductive organs, gastrointestinal tract inflammation, synovitis, or lymphocytic infiltration of the kidney, salivary gland, lacrimal gland, or stomach.
In one embodiment, a cell isolated from a mouse of any of the present embodiments is provided. In some aspects, the cell is an immune cell. In some aspects, the cell is a T cell.
In one embodiment, methods are provided for screening at least one candidate agent in a mouse of the present embodiments, the methods comprising administering one or more candidate agent to the mouse. In some aspects, the methods further comprise screening the at least one candidate agent in a mouse comprising (i) a homozygous wild-type Ctla4 gene and (ii) a homozygous loss-of-function of a Pdcd1 gene; a mouse comprising (i) a homozygous wild-type Ctla4 gene and (ii) a homozygous wild-type Pdcd1 gene; a mouse comprising (i) a homozygous wild-type Ctla4 gene and (ii) a heterozygous loss-of-function allele of a Pdcd1 gene; a mouse comprising (i) a heterozygous loss-of-function allele of a Ctla4 gene and (ii) a homozygous wild-type Pdcd1 gene; and/or a mouse comprising (i) a heterozygous loss-of-function allele of a Ctla4 gene and (ii) a heterozygous loss-of-function of a Pdcd1 gene; and/or a mouse comprising (i) a heterozygous loss-of-function allele of a Ctla4 gene and (ii) a homozygous loss-of-function allele of a Pdcd1 gene.
In some aspects, the at least one candidate therapeutic agent is screened for its ability to mitigate an immune-related adverse event or immune-related condition. In some aspects, mitigating an immune-related adverse event or immune-related condition is further defined as preventing the development of the immune-related adverse event or immune-related condition. In some aspects, mitigating an immune-related adverse event or immune-related condition is further defined as decreasing the severity of the immune-related adverse event or immune-related condition. In some aspects, mitigating an immune-related adverse event or immune-related condition is further defined as mitigating the mortality resulting from Ctla4 haploinsufficiency. In some aspects, mitigating an immune-related adverse event or immune-related condition is further defined as mitigating a systemic immune-related adverse event or immune-related condition. In some aspects, mitigating an autoimmunity is further defined as mitigating organ- or tissue-specific autoimmunity. In some aspects, mitigating an immune-related adverse event or immune-related condition is further defined as decreasing the severity of the immune-related adverse event or immune-related condition. In some aspects, mitigating an immune-related adverse event or immune-related condition is further defined as decreasing the frequency at which the immune-related adverse event or immune-related condition manifests in the population of mice. In some aspects, mitigating an immune-related adverse event or immune-related condition is further defined as slowing the development, or time to onset, of the immune-related adverse event or immune-related condition. In some aspects, screening a candidate therapeutic agent is defined as testing the efficacy of the candidate therapeutic agent.
In some aspects, the immune-related adverse event or immune-related condition is inflammation, such as, for example, acute inflammation or chronic inflammation. In some aspects, the immune-related adverse event or immune-related condition is autoimmunity or an autoimmune condition. In some aspects, the immune-related adverse event or immune-related condition comprises an immune-related adverse event or immune-related condition that mimics an immune-related adverse event or an autoimmunity induced by a checkpoint blockade therapy in humans. In certain aspects, the immune-related adverse event or immune-related condition is cardiac autoimmunity or pancreatic autoimmunity. In one aspect, the cardiac autoimmunity is myocarditis, such as, for example, fulminant myocarditis. In one aspect, the pancreatic autoimmunity is insulin-dependent diabetes mellitus.
In some aspects, the candidate agent is a CTLA4-immunoglobulin fusion protein (e.g., abatacept or a murine version thereof), a steroid, an agent that depletes a specific population of immune cells (e.g., anti-CD4 antibody to deplete CD4 T cells or anti-CD20 monoclonal antibody (e.g., rituximab) to deplete B cells), a cytokine modulating agent (e.g., toclizumab or a murine version thereof), or an immunosuppressive agent. In some aspects, the candidate agent is an anti-cancer therapy (e.g., chemotherapy, radiation, surgery, kinase inhibitors, immunotherapies, anti-TIM3, anti-OX40, oncolytic viruses, bispecific antibodies) and the method is screening for additional adverse events that occur in mice suffering from autoimmunity that mimics an immune-related adverse event. The screening may identify therapeutic agents, that when combined with immune checkpoint blockade, have an unfavorable risk profile for the development of autoimmunity or immune-related adverse events. In some aspects, the candidate agent is a pathogen (e.g., commensal or infectious), stress, an injury, and/or a diet. In some aspects, the candidate agent is a tumor cell, such as a syngeneic tumor cell (e.g., B16 melanoma, MC38 colon carcinoma, Lewis lung carcinoma). The tumor cell may be engrafted into the mouse and the effect of the resulting tumor on the immune-related adverse events may be characterized. Tumor properties tested may include total tumor burden, tumor lysis resulting from therapy, release of tumor-associated antigens (e.g., injection of irradiated tumor cells as a tumor immunization), or specific properties of the tumor (e.g., specific mutations or activity of specific genes).
As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used herein “another” may mean at least a second or more.
Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
T cell activation is tightly regulated via a wide range of mechanisms including negative co-stimulation, but the extent to which individual molecular mediators functionally interact remains unclear. CTLA4 and PD-1 are key negative regulators of T cell activation that utilize distinct molecular and cellular mechanisms. CTLA4 and PD-1 are both T cell negative costimulatory molecules whose function is to attenuate T cell activity. These molecules utilize distinct molecular mechanisms to carry out these functions. These molecular mechanisms are mediated by largely distinct cell signaling pathways (Wei et al., 2018). The primary function of CTLA4 is to compete for binding of B7 ligands (B7-1/CD80, B7-2/CD86), which leads to a reduction in CD28 positive costimulation and downstream PI3K/AKT signaling. The primary function of PD-1 is to inhibit proximal T cell receptor signaling upon binding to its ligands PD-L1/PD-L2; however, inhibition of CD28 signaling has also been reported as a significant function of PD-1 (Hui et al., 2017). Antibody-mediated blockade inhibits these functions by preventing ligand binding. This loss of signaling capacity and the consequent downstream biological events can be modeled by genetic means through the combination of loss of function alleles of Ctla4 and Pdcd1.
The inventors sought to understand whether regulatory mechanisms imposed by CTLA4 and PD-1 are functionally independent or dependent and found evidence of genetic interaction between Ctla4 and Pdcd1 (encoding PD-1) in mice. Mono-allelic loss of Ctla4 in the context of complete genetic absence of Pdcd1 led to death in approximately half of mice. Mortality was caused by autoimmunity in multiple tissues, including pancreas and heart (see, e.g.,
This animal model will enable investigation into disease etiology and identification of factors that modulate the generation of immune related adverse events. This animal model develops autoimmunity in the heart and pancreas (as well as other organs), which is important because fatal myocarditis (inflammation of the heart) and type I diabetes (autoimmune destruction of the pancreas) are two types of rare but very serious complications associated with combination anti-CTLA4 and anti-PD-1 therapy in human patients. This model also appears to be able to model other types of autoimmune adverse events (e.g. gastrointestional) that are associated with checkpoint blockade. The transgenic mouse model described can be used as a model of combination anti-CTLA4 plus anti-PD-1 immune checkpoint blockade (i.e., treatment of monoclonal antibodies targeting T cell costimulatory receptors CTLA4 and PD-1). Genetic loss of PD-1 and single copy loss of CTLA4 models this therapy in an analogous scenario in which negative costimulatory activity is reduced and modeled. Thus, this genetic model that modulates CTLA4 and PD-1 recapitulates the phenomena of immune-related adverse events due to checkpoint blockade therapy. This is particularly notable because there are currently no animal models that faithfully recapitulate the adverse events associated with checkpoint blockade therapy. Combination anti-CTLA4 and anti-PD-1 checkpoint blockade therapy is currently approved for the treatment of melanoma and renal cell carcinoma (along with over 250 on-going clinical trials).
In addition, this mouse strain background is C57BL6/J, which is notable because of the widespread use of this background for tumor immunology studies and also because this background is normally very difficult to induce autoimmunity in, suggesting that this phenotype satisfies a high biological bar. Also, no exogenous antigens were introduced (e.g. transgenic, viral) and thus the antigens that are being recognized to drive this autoimmunity are self-antigens, as is presumed to be the case in the setting of patients that receive checkpoint blockade therapies. Furthermore, because multiple types of autoimmunity and immune-related disease etiologies arise in this animal model, this enables the investigation of the relationships between these diseases.
Thus, this mouse model can be used to understand how these adverse events are induced as well as to test the efficacy of therapies that aim to mitigate such adverse events and autoimmunity. Such investigation is likely necessary to design next-generation immunotherapies that retain therapeutic efficacy and reduce adverse events, particularly induction of rare, very serious autoimmunity.
A genetic interaction between the T cell negative costimulatory genes Ctla4 and Pdcd1 (encoding PD-1) is identified herein. This genetic interaction manifests as conditional haploinsufficiency of Ctla4 in the context of complete absence of Pdcd1, which leads to fatal systemic autoimmunity. From a fundamental perspective, this observation supports a threshold model of T cell activation in which multiple sources of T-cell receptor (TCR) signal perturbation can compound to result in aberrant T cell activation. This indicates that CTLA4 and PD-1 regulatory signals are functionally integrated and together provide a critical buffering system to restrain T cell activation. At the molecular level, decreases in the combined gene dosage of Pdcd1 and Ctla4 may limit the overall ability to attenuate T cell activation in a cell intrinsic manner.
In addition to the significant insights into basic mechanisms of T cell activation, these findings have notable clinical implications in the context of cancer immune checkpoint blockade. Combination anti-CTLA4 plus anti-PD-1 therapy is effective in multiple tumor types, including advanced melanoma and renal cell carcinoma. Anti-CTLA4 and anti-PD-1 immune checkpoint blockade are known to utilize distinct cellular mechanisms (Das et al., 2015; Wei et al., 2017). Taken in the context of the present findings, these distinct cellular mechanisms likely interact functionally, which may in part explain the enhanced efficacy of combination therapy versus monotherapies (Curran et al., 2010; Postow et al., 2015; Wolchok et al., 2013). Even more relevant to the present findings, immune checkpoint blockade therapy can induce serious immune-related adverse events as well as bona fide autoimmunity, such as myocarditis and type I diabetes, in rare instances. Ctla4+/− Pdcd1−/− mice provide a preclinical animal model with which to study autoimmunity induced by loss of CTLA4 and PD-1 signaling. This is particularly notable given that autoimmunity observed in this model closely reflect the myocarditis and type I diabetes that can arise following combination anti-CTLA4 plus anti-PD-1 checkpoint blockade therapy in human patients. Further mechanistic understanding provides the potential to distinguish and specifically modulate aspects of the immunological response that mediate efficacy and adverse events of immune checkpoint blockade therapy.
Interestingly, the autoimmunity that develops in Ctla4+/− Pdcd1−/− mice recurrently manifests in specific anatomical sites. Why particular tissue sites are more sensitive to autoimmunity induced by loss of PD-1 and CTLA4 signaling remains a critical open question. It is possible that tissue specific antigens from these sites render them particularly liable to autoimmune recognition in the absence of negative co-stimulation. Alternatively, it is possible that tissue sensitivity is due to functional differences between tissue-specific Treg populations that have been previously observed (Legoux et al., 2015).
Notably, heterozygous germ line loss of function alleles of CTLA4 lead to immune dysregulation with highly variable clinical presentation (Kuehn et al., 2014; Schubert et al., 2014). This indicates that single copy loss of CTLA4 in humans is pathogenic and furthermore, is strongly suggestive of genetic interaction with other genetic and/or environmental factors. It is also possible that single copy loss of CTLA4 in humans or in mice (in the absence of PD-1) lowers the T cell activation threshold to the level at which tonic TCR signaling can reach, and thus stochastic processes may explain the variance in clinical presentation in CTLA4 deficient humans and mice. An outstanding question is the extent to which other T cell costimulatory molecules or molecules involved in their function genetically interact with Pdcd1 and Ctla4. For example, patients harboring loss of function alleles of LRBA, an important regulator of CTLA4 trafficking, present with similar autoimmune phenotypes as patients harboring loss of function CTLA4 (Besnard et al., 2018; Hou et al., 2017).
In addition to the key finding of genetic interaction between CTLA4 and PD-1, the present findings also suggest that simultaneous genetic deficiency of Ctla4 and Pdcd1 is embryonic lethal. This is surprising given that αβ T cells do not emerge until after birth (Havran and Allison, 1988). The mechanism through which this occurs remains unclear, however there are two main possibilities, both of which are quite intriguing. The first possibility is that CTLA4 and PD-1 restrict activation of yδ T cells, which emerge as early as E14 during embryonic development. The second possibility is that CTLA4 and PD-1 may have as yet unidentified non-immunological functions during development.
In conclusion, the present findings reveal genetic interaction between Ctla4 and Pdcd1. This provides definitive evidence for functional interaction between the regulatory mechanisms of CTLA4 and PD-1. Furthermore, this provides a robust animal to model immune-related adverse events induced by combination anti-CTLA4 plus anti-PD-1 immune checkpoint blockade therapy.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Mice. Ctla4tm1All mice (Chambers et al., 1997) were bred to Pdcd1 knockout mice (Pdcd1tm1.1Shr) (Keir et al., 2007), which were purchased from The Jackson Laboratory (021157). Pdcd1 knockout mice were backcrossed once to C57BL/6J prior to this cross. Resulting F1 Ctla4+/− Pdcd1+/− mice were intercrossed to produce all possible combinations of wild-type and mutant alleles of the two genes. This first breeding scheme specifically utilized F1 mice derived from the cross of Ctla4+/− (which are wild-type for Pdcd1) and Pdcd1−/− mice (which are wild-type for Ctla4). This breeding scheme ensures that mutant alleles of Ctla4 and Pdcd1 in F1 mice are in trans, and thus the recombination frequency can be calculated (
To verify findings from the first breeding scheme, a second related breeding scheme was utilized. Importantly, this breeding approach utilized different genotypes, the mutant alleles could either be in cis or trans, and the approach would generate Ctla4+/− Pdcd1−/− (experimental) and Ctla4+/+ Pdcd1−/− (control littermates) in a 1:1 ratio. This allows for the generation of many more Ctla4+/− Pdcd1−/− mice than in the initial breeding approach. Specifically, male Ctla4+/− Pdcd1−/− and female Ctla4+/+ Pdcd1−/− were bred. Female Ctla4+/+ Pdcd1−/− were used to eliminate the possibility that the autoimmunity observed in Ctla4+/− Pdcd1−/− might affect fetal-maternal tolerance or the ability to produce viable litters.
For the generation of survival curves, events were defined as either death (i.e. mice found dead) or identification of mice by veterinary staff as requiring euthanasia (e.g. due to lethargy, moribund, dyspnea). For mice identified as requiring euthanasia, the date of death was defined as the day the mouse was flagged by veterinary staff. Animal phenotypes associated with mortality were identified and reported by veterinary staff. Mice utilized for breeding were censored from survival analyses at the time that they were utilized for this purpose.
All mice were housed at The University of Texas MD Anderson Cancer Center South Campus Vivarium, an AAALAC-accredited specific pathogen-free animal facility. All experiments were performed in accordance with The University of Texas MD Anderson Cancer Center Institutional Animal Care and Use Committee (IACUC) guidelines.
Genotyping. Genomic DNA was isolated using Direct-to-PCR digest mix and polymerase chain reaction (PCR) based genotyping was performed for Ctla4 and Pdcd1 knockout mice. Primers are provided in Table 1. Ctla4tm1All mice were genotyped as previously described (Chambers et al., 1997). The expected band sizes for the Ctla4 wild-type and mutant alleles are ˜75 and ˜150 bp, respectively. Pdcd1 knockout mice were genotyped as previously described (Keir et al., 2007). The expected band sizes for the Pdcd1 wild-type and mutant alleles are 418 and 350 bp, respectively.
SNP typing. Crude genomic DNA lysate was submitted to The University of Texas MD Anderson Cancer Center Laboratory Animal Genetics Services core facility for SNP-typing using a 100-marker panel. For the purpose of determining whether genetic variants associate with autoimmunity, ‘unaffected’ mice were defined as mice that did not manifest any symptoms or die within 6 months of age and ‘affected’ mice were defined as class I mice that manifested symptoms and succumbed to disease.
Pathology analyses. Animal necropsies were performed by personnel in The University of Texas MD Anderson Cancer Center veterinary medical histology laboratory or in the Allison laboratory. Automated serum chemistry analysis using a Cobas Integra 400Plus (Roche Diagnostics, Risch-Rotkreuz, Switzerland) was performed on a blood sample collected at euthanasia. Formalin-fixed tissues were processed routinely into paraffin blocks, sectioned at 5 microns, and stained with hematoxylin and eosin. Additional sections were used for immunohistochemical (IHC) staining of particular tissues of interest, using an antibody directed against CD3 (ab16669, Abcam, Cambridge, Mass.), followed by secondary reagents for chromogenic detection (Bond Polymer Refine Detection system, DS9800, Leica, Buffalo Grove, Ill.). Stained sections were examined by a veterinary pathologist using a Leica DM2500 microscope with Leica DFC495 camera and Leica Application Suite v4.12 software. Histologic changes were scored using a semi-quantitative scale, with 0=no lesion to 4=severe lesion.
Flow cytometry. Single cell suspensions from lymph nodes were prepared by mashing pooled inguinal, axillary, and brachial lymph nodes through a 70 um filter using the back of a plastic syringe into RPMI-1640 supplemented with 10% FBS and 1% Penicillin Streptomycin. A 96-well flat bottom plate was coated 200 ul per well of 1 ug/ml anti-CD3E and 2 ug/ml anti-CD28 in PBS overnight at 4° C. the previous night. Cells were then stained with CellTrace Violet Proliferation kit per the manufactures protocol (Invitrogen, C34557). Triplicates of each sample were plated 106 cells/mL per well in 200 ul of RPMI-1640 supplemented with 10% FBS, sodium pyruvate, 0.1% b-ME, and P/S and incubated at 37° C. for 46 hours. Cells were then transferred to a U-bottom 96-well plate and washed twice with FACS buffer and incubated with 2% of each bovine, murine, rat, hamster, and rabbit serum PBS with 25 mg/mL 2.4G2 antibody at 4° C. for 10 min prior to surface staining with an antibody cocktail at 4° C. for 30 min in a 50 mL volume. Cells were washed twice with FACS buffer then fixed and permeabilized using the FoxP3 fix and permeabilization kit according to manufacturer's protocol (eBioscience). Cells were subsequently stained with an intracellular antibody cocktail at room temperature for 30 min. Cells were then washed twice with Foxp3 permeabilization buffer, then twice with FACS buffer, and analyzed on a LSRII (BD).
For surface stain (restim) the following antibodies were used: LIVE/DEAD™ Fixable Blue Dead Cell Stain L23105 (ThermoFisher); BV786 Hamster Anti-Mouse CD3e (clone 145-2C11, 564379 (BD)); Brilliant Violet 605 anti-mouse TCR 13 chain Antibody (clone)H57-597, 109241(BioLegend)); Brilliant Violet 650 anti-mouse CD19 Antibody (clone 6D5, 115541 (BioLegend)); FITC Anti-mouse CD4 Antibody (clone RM4.5, 11-0042-82 (ebio)); PE anti-mouse CD152 Antibody (clone UC10-4B9, 106306 (BioLegend)); PE Armenian Hamster IgG Isotype Ctrl Antibody (clone HTK888, 400908 (Biolegend); APC Anti-mouse CD8a Antibody (clone 53-6.7, 17-0081-82 (ebio)); and Alexa Fluor 700 Anti-mouse CD45.2 Antibody, (clone 104, 56-0454-82 (ebio)). For IC stain (restim) the following antibodies were used: BV786 Hamster Anti-Mouse CD3e (clone 145-2C11, 564379 (BD)); Brilliant Violet 605 anti-mouse TCR (3 chain Antibody (clone H57-597, 109241(BioLegend)); FITC Anti-mouse CD4 Antibody (clone RM4.5, 11-0042-82 (ebio)); PE anti-mouse CD152 (CTLA-4) Antibody (clone UC10-4B9, 106306 (BioLegend)); PE Armenian Hamster IgG Isotype CTLA-4 Ctrl Antibody (clone HTK888, 400908 (BioLegend)); and APC Anti-mouse CD8a Antibody (clone 53-6.7, 17-0081-82 (ebio)).
Luminex cytokine and chemokine assessment. Serum was collected from Ctla4+/− Pdcd1−/− and control littermate mice (including both Ctla4+/+ Pdcd1−/− mice and mice competent for both CTLA4 and PD-1 such as Ctla4+/− Pdcd1−/− mice) from both breeding schemes described above. Briefly, blood was collected by terminal cardiac puncture, allowed to coagulate at room temperature, centrifuged at 8,000 g for 10 minutes, supernatant serum collected and snap frozen in liquid nitrogen prior to storage at −80 degrees Celsius. Serum levels of antibodies cytokine and chemokine were assessed using the Cytokine & Chemokine 36-plex Mouse ProcartPlex luminex assay (ThermoFisher Scientific) per manufacturer's protocol. All samples were analyzed in parallel in a single batch for each respective analysis. Serum samples were diluted 1:10,000 for analysis of serum antibody levels.
Reverse phase proteomic array analysis. Lymph nodes from 16-day-old Ctla4 knockout and littermate control mice were snap frozen for subsequent analysis. Tissue samples were lysed in 1% Triton X-100, 50 mM HEPES, pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM Na pyrophosphate, 1 mM Na3VO4, 10% glycerol, with freshly added protease inhibitors (Roche, 05056489001) and phosphatase inhibitors (Roche, 04906837001) and homogenized using a Precellys homogenizer (Bertin Instruments). Samples were diluted in sample buffer (10% glycerol, 2% SDS, 62.5 mM Tris-HCl, BME, pH 6.8) prior to array printing and analysis. Signal was quantified using Array-Pro Analyzer software (MediaCybernetics) and normalized using a “Supercurve fitting” approach developed at MD Anderson Cancer Center for the RPPA Core Facility. Normalized linear values were analyzed in Excel (Microsoft) using two-tailed T-test assuming unequal variance and plotted using Prism 6.0 (GraphPad).
Nanostring mRNA analysis. Lymph nodes were dissected from Ctla4 knockout and littermate control mice. RNA was extracted from lymph nodes using (Qiagen). 100 ng RNA was analyzed using the Mouse Immunology Code set panel on the nCounter platform (Nanostring). Values were normalized on a per sample basis using housekeeping genes within the panel. Heat maps of Nanostring gene expression and RPPA proteomic data were generated in R utilizing a Pearson distance matrix and Ward's minimum variance method. Only differentially expressed genes, defined by a false discovery rate of 5% with a one-way ANOVA comparison between genotypes, were plotted.
Statistics. Statistical analyses were performed in Prism 7.0 or 8.0 (GraphPad Software, San Diego, Calif.), unless otherwise noted. Normalized linear values of RPPA data were analyzed in Excel (Microsoft) using two-tailed T-test assuming unequal variance and plotted.
To test whether there is a genetic interaction between Ctla4 and Pdcd1, Ctla4 and Pdcd1 (encoding PD-1) knockout transgenic mice were crossed. Murine Ctla4 and Pdcd1 are genetically linked with a genetic distance of 16.89 cM based on estimations from the Mouse Phenome Database (Bogue et al., 2018). A heterozygous intercross-breeding scheme (see Materials and Methods) was used that allowed for the generation of all possible permutations of mutant alleles from a single cross and for estimation of the observed recombination frequency between Ctla4 and Pdcd1 at 17.03 cM. Surprisingly, approximately 50% of Ctla4+/− Pdcd1−/− spontaneously died within 3 months of age (
Of particular interest was the surprising spontaneous death of Ctla4+/− Pdcd1−/− mice as this provides strong evidence of genetic interaction. To further confirm this observation, Ctla4+/− Pdcd1−/− and Ctla4+/+ Pdcd1−/− littermate mice were generated using a different breeding scheme (see Materials and Methods). This second approach yielded remarkably similar findings with approximately 50% of Ctla4+/− Pdcd1−/− mice spontaneously dying while no deaths were observed in littermate Ctla4+/+ Pdcd1−/− mice (
Together these observations indicate that Pdcd1 and Ctla4 exhibit strong genetic interaction. Specifically, there is a dramatic Ctla4 conditional haploinsufficiency, which manifests only in the context of genetic deletion of Pdcd1 and leads to spontaneous deaths (
Given that only 50% of Ctla4+/− Pdcd1−/− mice die, it is likely that additional genetic or environmental factors modulate the penetrance of Ctla4 conditional haploinsufficiency. Whether subtle genetic differences could underlie this dichotomy was investigated. All tested mice were 97-100% C57BL6/J based on a 100-marker single nucleotide polymorphism typing panel, with no associations between any segregating alleles with phenotypic manifestation were observed (
It was then determined whether Ctla4+/− Pdcd1−/− mice that did not spontaneously die and reached the age of the survival plateau (approximately 6 months) harbored subtle autoimmunity, which is biologically relevant but not sufficient to cause death. In contrast, the total body weights of affected Ctla4+/− Pdcd1−/− mice were significantly reduced compared to either phenotypically normal Ctla4+/− Pdcd1−/− or Ctla4+/+ Pdcd1−/− mice (
These data support a model in which environmental factors modulate the penetrance and development of fatal phenotypes in Ctla4+/− Pdcd1−/− mice. From a fundamental perspective, this observation supports a threshold model of T cell activation in which multiple sources of TCR signal perturbation are integrated to regulate T cell activation. In the case of Ctla4+/− Pdcd1−/− mice, the threshold for activation is significantly lowered such that additional subtle inputs, which are normally buffered, are sufficient to induce aberrant T cell activation and autoimmunity. A prediction of this model is that T cells derived from Ctla4+/− Pdcd1−/− mice have decreased levels of CTLA-4 compared to T cells derived from Ctla4+/+ Pdcd1−/− mice. To confirm that single copy loss of Ctla4 leads to a decrease in available CTLA-4 protein, we assessed total CTLA-4 protein expression in activated T cells from Ctla4+/− Pdcd1−/− and littermate control Ctla4+/+ Pdcd1−/− mice. Notably, flow cytometry analysis of in vitro stimulated T cells are suggestive of lower levels of CTLA-4 protein in T cells derived from Ctla4+/− Pdcd1−/− mice compared to T cells derived from Ctla4+/+ Pdcd1−/− mice (
Taken in the context of prior findings that Ctla4+/− mice do not display any haploinsufficiency at either the organismal or cellular level, these findings indicate that PD-1 negative co-stimulation is sufficient to functionally buffer mono-allelic loss of Ctla4. Consistent with this notion, few differences were observed in transcriptional and proteomic analyses of lymph nodes from Ctla4+/+ and Ctla4+/− mice where dramatic changes were observed in Ctla4−/− mice (
Interestingly, these results contrast the findings from mass cytometry profiling of similar tissues from Ctla4+/+ and Ctla4+/− mice (with no perturbation in Pdcd1), in which no differences in cellular phenotype or frequency were detected. This suggests that at least in homogeneous inbred murine strains, additional regulatory molecular mechanisms can buffer against perturbations in signaling caused by single copy loss of Ctla4. However, in the context of additional perturbations, such as genetic loss of PD-1, functional defects in T cell regulation due to mono-allelic Ctla4 can manifest due to a loss of buffering capacity.
It was next sought to understand the cause of death of Ctla4+/− Pdcd1−/− mice and investigate whether specific tissues were affected. In addition, it was sought to understand whether particular cell types mediated disease etiology. To address these questions, 42 tissues from Ctla4+/+ Pdcd1−/− and Ctla4+/− Pdcd1−/− mice were histologically analyzed (see Materials and Methods). Inflammation was observed in multiple tissues including heart, pancreas, lung, liver, and gastrointestional tract. Specific pathologic observations include lymphocytic myocarditis, endarteritis, pulmonary vasculitis, and lymphocytic pancreatitis. Of the most dramatic histological findings, significant immune infiltrate was observed in heart and pancreatic tissues of Ctla4+/− Pdcd1−/− mice (
To better interrogate nature of the myocardial infiltrates, detailed H&E histological analyses of a larger cohort of mice as well as immunohistochemical staining of similar tissue samples for T cells (utilizing CD3 as a pan-T cell marker) was performed. Myocarditis consisted of significant T cell infiltration in Ctla4+/− Pdcd1−/− mice. Histological analyses also were suggestive of infiltration of other immune populations such as macrophages. These data suggest that the myocarditis seen in the Ctla4+/− Pdcd1−/− mice is histologically similar to patients with ICI-associated myocarditis. The results of a range of histological analyses are summarized below in Tables 3-4.
Severe atrophy of adipose tissue associated with a wide range of anatomical sites including lung and subcutaneous (skin) was also observed (
These findings are further notable given that the C57BL6/J inbred strain of mice is highly resistant to the development of autoimmunity. For example, consistent with the findings here, mice deficient for PD-1 develop more severe autoimmunity on a Balb/c background compared to a C57BL6/J background (Nishimura et al., 1999; Nishimura et al., 2001). It is also important to note that the autoimmunity that develops due to genetic loss of PD-1 in aged mice is primarily mediated by auto-antibodies. However, antibody levels were not elevated in Ctla4+/− Pdcd1−/− mice (
Of note, these findings bear striking resemblance to the autoimmunity and other immune related adverse events (irAEs) associated with combination anti-CTLA4 plus anti-PD-1 immune checkpoint blockade therapy (e.g., ipilimumab plus nivolumab) (Sznol et al., 2017). In particular, the severe autoimmunity observed in pancreatic and cardiac tissue observed in Ctla4+/− Pdcd1−/− mice appears to be analogous to the fulminant myocarditis and insulin-dependent diabetes mellitus that are rare but very serious adverse events associated with therapeutic blockade of CTLA-4 and PD-1 (Barroso-Sousa et al., 2018; Johnson et al., 2016; Moslehi et al., 2018). Supportive of the notion that Ctla4+/− Pdcd1−/− mice closely recapitulates this biology, therapy associated myocarditis and diabetes appear to be directly T cell mediated.
It was then sought to gain insight into the antigen specificity of the T cells that underlies the systemic autoimmunity induced by conditional haploinsufficiency of Ctla4, and in particular, whether particular tissue antigens were being recurrently recognized. To explore this possibility, TCR sequencing was performed on lymph node, heart, and pancreatic tissues from Ctla4+/+ Pdcd1−/− and Ctla4+/− Pdcd1−/− mice (see Materials and Methods). Interestingly, no significant changes in T cell clonality were observed between Ctla4+/+ Pdcd1−/− and Ctla4+/− Pdcd1−/− mice (
To assess whether autoimmunity due to conditional haploinsufficiency of Ctla4 in the absence of PD-1 arises solely due to defects in peripheral tolerance, or also defects in central tolerance, thymic development in Ctla4+/− Pdcd1−/− and littermate Ctla4+/+ Pdcd1−/− mice were characterized. Mono-allelic loss of Ctla4 did not affect thymocyte composition, consistent with prior reports that CTLA-4 does not play a critical role during thymic development (Chambers et al., 1997; Wei et al., 2019). Consistent with these observations in lymph node derived T cells, thymic-derived Tregs (newly generated and recirculating) derived from Ctla4+/− Pdcd1−/− mice expressed decreased CTLA-4 protein. Together these data indicate that Ctla4 haploinsuffiency leads to a defect in peripheral tolerance rather than a defect in central tolerance.
Finally, the molecular basis of the genetic interaction between Ctla4 and Pdcd1 was investigated. Single copy loss of Ctla4 was hypothesized to lead to subtle changes in signaling and transcriptional outputs that in the context of an otherwise wild-type condition, do not modulate T cell activity or phenotype due to robust buffering within T cell activation signaling pathways. However, in the additional absence of PD-1, or perhaps other negative costimulatory molecules, subtle molecular defects due to Ctla4 haploinsufficiency can manifest overtly. To explore this possibility, we utilized reverse phase proteomic analysis (RPPA) to probe the expression of 238 protein targets in lymph node tissue derived from wild-type, heterozygous, and homozygous Ctla4 knockout mice. This RPPA panel included an array of signaling molecules and phosphorylated epitopes, and thus is well suited to detect changes in canonical signaling pathways.
As expected, proteins associated with proliferation pathways were highly upregulated in Ctla4−/− mice compared to littermate controls, consistent with the lymphoproliferative phenotype of Ctla4 knockout mice. This included significant increases in CDK1, p-Rb, p-S6, p-CHK1, and p-STAT3, accompanied by down-regulation of p21 (
All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
This application claims the benefit of U.S. Provisional Patent Application No. 62/729,965, filed Sep. 11, 2018, the entirety of which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/050551 | 9/11/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62729965 | Sep 2018 | US |
