A High-Throughput Method for Characterizing Novel Chimeric Antigen Receptors in Jurkat Cells.

Chimeric antigen receptor (CAR) development involves extensive empirical characterization of antigen-binding domain (ABD)/CAR constructs for clinical suitability. Here, we present a cost-efficient and rapid method for evaluating CARs in human Jurkat T cells. Using a modular CAR plasmid, a highly efficient ABD cloning strategy, plasmid electroporation, short-term co-culture, and flow-cytometric detection of CD69, this assay (referred to as CAR-J) evaluates sensitivity and specificity for ABDs. Assessing 16 novel anti-CD22 single-chain variable fragments derived from mouse monoclonal antibodies, CAR-J stratified constructs by response magnitude to CD22-expressing target cells. We also characterized 5 novel anti-EGFRvIII CARs for preclinical development, identifying candidates with varying tonic and target-specific activation characteristics. When evaluated in primary human T cells, tonic/auto-activating (without target cells) EGFRvIII-CARs induced target-independent proliferation, differentiation toward an effector phenotype, elevated activity against EGFRvIII-negative cells, and progressive loss of target-specific response upon in vitro re-challenge. These EGFRvIII CAR-T cells also showed anti-tumor activity in xenografted mice. In summary, CAR-J represents a straightforward method for high-throughput assessment of CAR constructs as genuine cell-associated antigen receptors that is particularly useful for generating large specificity datasets as well as potential downstream CAR optimization.

Llama peripheral B-cell populations producing conventional and heavy chain-only IgG subtypes are phenotypically indistinguishable but immunogenetically distinct.

Camelid ungulates produce homodimeric heavy chain-only antibodies (HCAbs) in addition to conventional antibodies consisting of paired heavy and light chains. In the llama, HCAbs are made up by at least two subclasses (long-hinge IgG2b and short-hinge IgG2c HCAbs vs. conventional heterotetrameric IgG1s). Here, we generated murine monoclonal antibodies (mAbs) specific for the hinge-CH2 boundary of llama IgG2b (mAb 1C10) and the Fc of llama IgG2c HCAbs (mAb 5E4). Flow cytometric analysis of llama peripheral blood lymphocytes revealed that IgG1+, IgG2b+ and IgG2c+ B cells could be distinguished using mAbs 1C10/5E4 but had equivalent expression of three other cell-surface markers. MiSeq sequencing of the peripheral B cell repertoires of three llamas showed that (i) IgG2b and IgG2c HCAbs were present in similar proportions in the repertoire, (ii) a subset of IgG2b and IgG2c HCAbs, but not IgG1s, entirely lacked a hinge exon and showed direct VHH-CH2 splicing; these "hingeless" HCAbs were clonally expanded, somatically mutated and derived from hinged HCAb precursors, (iii) substantial repertoire overlap existed between IgG subclasses, especially between IgG2b and IgG2c HCAbs, (iv) the complementarity-determining region (CDR)-H3 length distributions of IgG2b and IgG2c HCAbs were broader and biased towards longer lengths compared with IgG1s due to increased N-nucleotide addition, (v) IgG2b and IgG2c HCAbs used a more restricted set of IGHV genes compared with IgG1s, and (vi) IgG2b and IgG2c HCAbs had elevated somatic mutations rates of both CDRs and framework regions (FRs) compared with IgG1s, especially of CDR-H1 and FR3. The distinct molecular features of llama IgG1, IgG2b and IgG2c antibodies imply that these subclasses may have divergent immunological functions and suggest that specific mechanisms operate to diversify HCAb repertoires in the absence of a light chain.

Deletion of a Yci1 Domain Protein of Candida albicans Allows Homothallic Mating in MTL Heterozygous Cells.

UNLABELLED: It has been proposed that the ancestral fungus was mating competent and homothallic. However, many mating-competent fungi were initially classified as asexual because their mating capacity was hidden behind layers of regulation. For efficient in vitro mating, the essentially obligate diploid ascomycete pathogen Candida albicans has to change its mating type locus from heterozygous MTLa/α to homozygous MTLa/a or MTLα/α and then undergo an environmentally controlled epigenetic switch to the mating-competent opaque form. These requirements greatly reduce the potential for C. albicans mating. Deletion of the Yci1 domain gene OFR1 bypasses the need for C. albicans cells to change the mating type locus from heterozygous to homozygous prior to switching to the opaque form and mating and allows homothallic mating of MTL heterozygous strains. This bypass is carbon source dependent and does not occur when cells are grown on glucose. Transcriptional profiling of ofr1 mutant cells shows that in addition to regulating cell type and mating circuitry, Ofr1 is needed for proper regulation of histone and chitin biosynthesis gene expression. It appears that OFR1 is a key regulator in C. albicans and functions in part to maintain the cryptic mating phenotype of the pathogen. IMPORTANCE: Candida albicans is a human fungal pathogen with a recently discovered, highly cryptic mating ability. For efficient mating, it has to lose heterozygosity at its mating type locus. Then, MTL homozygous strains can undergo an epigenetic switch to an elongated yeast state, termed the opaque form, and become mating competent. This infrequent two-step process greatly reduces the potential for mating; few strains are MTL homozygous, and the opaque state is unstable at the temperature of the mammalian host. C. albicans has a complex mechanism for mating that appears designed to ensure that mating is infrequent. Here, we have characterized a new gene, opaque-formation regulator 1 (OFR1). Deleting the OFR1 gene allows MTL A: /α strains to mate efficiently with either mating type or even mate homothallically. It is possible that downregulating OFR1 in the host environment could allow mating in C. albicans by a route that does not involve MTL homozygosis.

Comparative xylose metabolism among the Ascomycetes C. albicans, S. stipitis and S. cerevisiae.

The ascomycetes Candida albicans, Saccharomyces cerevisiae and Scheffersomyces stipitis metabolize the pentose sugar xylose very differently. S. cerevisiae fails to grow on xylose, while C. albicans can grow, and S. stipitis can both grow and ferment xylose to ethanol. However, all three species contain highly similar genes that encode potential xylose reductases and xylitol dehydrogenases required to convert xylose to xylulose, and xylulose supports the growth of all three fungi. We have created C. albicans strains deleted for the xylose reductase gene GRE3, the xylitol dehydrogenase gene XYL2, as well as the gre3 xyl2 double mutant. As expected, all the mutant strains cannot grow on xylose, while the single gre3 mutant can grow on xylitol. The gre3 and xyl2 mutants are efficiently complemented by the XYL1 and XYL2 from S. stipitis. Intriguingly, the S. cerevisiae GRE3 gene can complement the Cagre3 mutant, while the ScSOR1 gene can complement the Caxyl2 mutant, showing that S. cerevisiae contains the enzymatic capacity for converting xylose to xylulose. In addition, the gre3 xyl2 double mutant of C. albicans is effectively rescued by the xylose isomerase (XI) gene of either Piromyces or Orpinomyces, suggesting that the XI provides an alternative to the missing oxido-reductase functions in the mutant required for the xylose-xylulose conversion. Overall this work suggests that C. albicans strains engineered to lack essential steps for xylose metabolism can provide a platform for the analysis of xylose metabolism enzymes from a variety of species, and confirms that S. cerevisiae has the genetic potential to convert xylose to xylulose, although non-engineered strains cannot proliferate on xylose as the sole carbon source.

Structurally unique interaction of RBD-like and PH domains is crucial for yeast pheromone signaling.

The Ste5 protein forms a scaffold that associates and regulates the components of the mitogen-activated protein (MAP) kinase cascade that controls mating-pheromone-mediated signaling in the yeast Saccharomyces cerevisiae. Although it is known that the MEK kinase of the pathway, Ste11, associates with Ste5, details of this interaction have not been established. We identified a Ras-binding-domain-like (RBL) region in the Ste11 protein that is required specifically for the kinase to function in the mating pathway. This module is structurally related to domains in other proteins that mediate Ras-MAP kinase kinase kinase associations; however, this RBL module does not interact with Ras, but instead binds the PH domain of the Ste5 scaffold. Structural and functional studies suggest that the key role of this PH domain is to mediate the Ste5-Ste11 interaction. Overall these two evolutionarily conserved modules interact with each other through a unique interface, and thus in the pheromone pathway the structural context of the RBL domain contribution to kinase activation has been shifted through a change of its interaction partner from Ras to a PH domain.

Reverse genetics in Candida albicans predicts ARF cycling is essential for drug resistance and virulence.

Candida albicans, the major fungal pathogen of humans, causes life-threatening infections in immunocompromised individuals. Due to limited available therapy options, this can frequently lead to therapy failure and emergence of drug resistance. To improve current treatment strategies, we have combined comprehensive chemical-genomic screening in Saccharomyces cerevisiae and validation in C. albicans with the goal of identifying compounds that can couple with the fungistatic drug fluconazole to make it fungicidal. Among the genes identified in the yeast screen, we found that only AGE3, which codes for an ADP-ribosylation factor GTPase activating effector protein, abrogates fluconazole tolerance in C. albicans. The age3 mutant was more sensitive to other sterols and cell wall inhibitors, including caspofungin. The deletion of AGE3 in drug resistant clinical isolates and in constitutively active calcineurin signaling mutants restored fluconazole sensitivity. We confirmed chemically the AGE3-dependent drug sensitivity by showing a potent fungicidal synergy between fluconazole and brefeldin A (an inhibitor of the guanine nucleotide exchange factor for ADP ribosylation factors) in wild type C. albicans as well as in drug resistant clinical isolates. Addition of calcineurin inhibitors to the fluconazole/brefeldin A combination only initially improved pathogen killing. Brefeldin A synergized with different drugs in non-albicans Candida species as well as Aspergillus fumigatus. Microarray studies showed that core transcriptional responses to two different drug classes are not significantly altered in age3 mutants. The therapeutic potential of inhibiting ARF activities was demonstrated by in vivo studies that showed age3 mutants are avirulent in wild type mice, attenuated in virulence in immunocompromised mice and that fluconazole treatment was significantly more efficacious when ARF signaling was genetically compromised. This work describes a new, widely conserved, broad-spectrum mechanism involved in fungal drug resistance and virulence and offers a potential route for single or improved combination therapies.

Chemogenomic profiling predicts antifungal synergies.

Chemotherapies, HIV infections, and treatments to block organ transplant rejection are creating a population of immunocompromised individuals at serious risk of systemic fungal infections. Since single-agent therapies are susceptible to failure due to either inherent or acquired resistance, alternative therapeutic approaches such as multi-agent therapies are needed. We have developed a bioinformatics-driven approach that efficiently predicts compound synergy for such combinatorial therapies. The approach uses chemogenomic profiles in order to identify compound profiles that have a statistically significant degree of similarity to a fluconazole profile. The compounds identified were then experimentally verified to be synergistic with fluconazole and with each other, in both Saccharomyces cerevisiae and the fungal pathogen Candida albicans. Our method is therefore capable of accurately predicting compound synergy to aid the development of combinatorial antifungal therapies.

Binding the atypical RA domain of Ste50p to the unfolded Opy2p cytoplasmic tail is essential for the high-osmolarity glycerol pathway.

Activation of the high-osmolarity glycerol (HOG) pathway for osmoregulation in the yeast Saccharomyces cerevisiae involves interaction of the adaptor Ste50p with the cytoplasmic tail of single-transmembrane protein Opy2p. We have determined the solution structure of the Ste50p-RA (Ras association) domain, and it shows an atypical RA fold lacking the beta1 and beta2 strands of the canonical motif. Although the core of the RA domain is fully functional in the pheromone response, an additional region is required for the HOG pathway activation. Two peptide motifs within the intrinsically disordered cytoplasmic tail of Opy2p defined by NMR spectroscopy physically interact with the Step50p-RA domain. These Opy2p-derived peptides bind overlapping regions of the Step50p-RA domain with similarly weak affinities, suggesting a multivalent interaction of these proteins as a crucial point of control of the HOG pathway. As well, overall selection of signaling pathways depends on functionally distinct regions of the Ste50p-RA domain, implicating this element in the control of global regulatory decisions.

Transcript profiling of a MAP kinase pathway in C. albicans.

In Candida albicans, a MAP kinase pathway has been implicated in aspects of controlling hyphal development. We have examined the transcription profile of cells deleted for the transcription factor Cph1p as well as Cst20p, Hst7p, and Cek1p, three upstream kinases potentially involved in Cph1p regulation. Deletion of any of these four elements does not block filament induction by serum and does not dramatically affect the transcription profile of the yeast-hyphal transition, but deletion of CPH1 delays filamentation. Over-expression of Cph1p by ADH1pt-CPH1 significantly enhances filamentation, suggesting that Cph1p is helpful but not essential for filament induction. Interestingly, the transcription profile of ADH1pt-CPH1 expressing cells under yeast conditions is similar to that of wild-type strains undergoing the yeast-hyphal transition. Finally, it appears that Cek1 and its regulators Hst7p and Cst20p may control the repression of genes such as CHT2 through a process independent of the Cph1p transcription factor.

A human-curated annotation of the Candida albicans genome.

Recent sequencing and assembly of the genome for the fungal pathogen Candida albicans used simple automated procedures for the identification of putative genes. We have reviewed the entire assembly, both by hand and with additional bioinformatic resources, to accurately map and describe 6,354 genes and to identify 246 genes whose original database entries contained sequencing errors (or possibly mutations) that affect their reading frame. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that might be targeted for antifungal therapy. We also observed that, compared to other fungi, the protein-coding sequences in the C. albicans genome are especially rich in short sequence repeats. Finally, our improved annotation permitted a detailed analysis of several multigene families, and comparative genomic studies showed that C. albicans has a far greater catabolic range, encoding respiratory Complex 1, several novel oxidoreductases and ketone body degrading enzymes, malonyl-CoA and enoyl-CoA carriers, several novel amino acid degrading enzymes, a variety of secreted catabolic lipases and proteases, and numerous transporters to assimilate the resulting nutrients. The results of these efforts will ensure that the Candida research community has uniform and comprehensive genomic information for medical research as well as for future diagnostic and therapeutic applications.

Transcription profiling of cyclic AMP signaling in Candida albicans.

We used transcription profiling in Candida albicans to investigate cellular regulation involving cAMP. We found that many genes require the adenylyl cyclase Cdc35p for proper expression. These include genes encoding ribosomal subunit proteins and RNA polymerase subunit proteins, suggesting that growth could be controlled in part by cAMP-mediated modulation of gene expression. Other genes influenced by loss of adenylyl cyclase are involved in metabolism, the cell wall, and stress response and include a group of genes of unknown function that are unique to C. albicans. The profiles generated by loss of the adenylyl cyclase regulator Ras1p and a downstream effector Efg1p were also examined. The loss of Ras1p function disturbs the expression of a subset of the genes regulated by adenylyl cyclase, suggesting both that the primary role of Ras1p in transcriptional regulation involves its influence on the function of Cdc35p and that there are Ras1p independent roles for Cdc35p. The transcription factor Efg1p is also needed for the expression of many genes; however, these genes are distinct from those modulated by Cdc35p with the exception of a class of hyphal-specific genes. Therefore transcription profiling establishes that cAMP plays a key role in the overall regulation of gene expression in C. albicans, and enhances our detailed understanding of the circuitry controlling this regulation.

Superoxide dismutases in Candida albicans: transcriptional regulation and functional characterization of the hyphal-induced SOD5 gene.

Superoxide dismutases (SOD) convert superoxide radicals into less damaging hydrogen peroxide. The opportunistic human pathogen Candida albicans is known to express CuZnSOD (SOD1) and MnSOD (SOD3) in the cytosol and MnSOD (SOD2) in the mitochondria. We identified three additional CuZn-containing superoxide dismutases, SOD4, SOD5, and SOD6, within the sequence of the C. albicans genome. The transcription of SOD5 was up-regulated during the yeast to hyphal transition of C. albicans, and SOD5 was induced when C. albicans cells were challenged with osmotic or with oxidative stresses. SOD5 transcription was also increased when cells were grown on nonfermentable substrates as the only carbon source. The Rim101p transcription factor was required for all inductions observed, whereas the Efg1p transcription factor was specifically needed for serum-modulated expression. Deletion of SOD5 produced a viable mutant strain that showed sensitivity to hydrogen peroxide when cells were grown in nutrient-limited conditions. Sod5p was found to be necessary for the virulence of C. albicans in a mouse model of infection. However, the sod5 mutant strain showed the same resistance to macrophage attack as its parental strain, suggesting that the loss of virulence in not due to an increased sensitivity to macrophage attack.

CDC42 is required for polarized growth in human pathogen Candida albicans.

Cdc42p is a member of the RAS superfamily of GTPases and plays an essential role in polarized growth in many eukaryotic cells. We cloned the Candida albicans CaCDC42 by functional complementation in Saccharomyces cerevisiae and analyzed its function in C. albicans. A double deletion of CaCDC42 was made in a C. albicans strain containing CaCDC42 under the control of the PCK1 promoter. When expression of the heterologous copy of CaCDC42 was repressed in this strain, the cells ceased proliferation. These arrested cells were large, round, and unbudded and contained predominantly two nuclei. The PCK1-mediated overexpression of wild-type CaCdc42p had no effect on cells. However, in cells overexpressing CaCdc42p containing the dominant-negative D118A substitution, proliferation was blocked and the arrested cells were large, round, unbudded, and multinucleated, similar to the phenotype of the cdc42 double-deletion strain. Cells overexpressing CaCdc42p containing the hyperactive G12V substitution also ceased proliferation in yeast growth medium; in this case the arrested cells were multinucleated and multibudded. An intact CAAX box is essential for the phenotypes associated with either CaCdc42p(G12V) or CaCdc42p(D118A) ectopic expression, suggesting that membrane attachment is involved in CaCdc42p function. In addition, the lethality caused by ectopic expression of CaCdc42p(G12V) was suppressed by deletion of CST20 but not by deletion of CaCLA4. CaCdc42p function was also examined under hypha-inducing conditions. Cdc42p depletion prior to hyphal induction trapped cells in a round, unbudded state, while depletion triggered at the same time as hyphal induction permitted the initiation of germ tubes that failed to be extended. Ectopic expression of either the G12V or D118A substitution protein modified hyphal formation in a CAAX box-dependent manner. Thus, CaCdc42p function appears important for polarized growth of both the yeast and hyphal forms of C. albicans.

Transcription profiling of Candida albicans cells undergoing the yeast-to-hyphal transition.

The ability of the pathogenic fungus Candida albicans to switch from a yeast to a hyphal morphology in response to external signals is implicated in its pathogenicity. We used glass DNA microarrays to investigate the transcription profiles of 6333 predicted ORFs in cells undergoing this transition and their responses to changes in temperature and culture medium. We have identified several genes whose transcriptional profiles are similar to those of known virulence factors that are modulated by the switch to hyphal growth caused by addition of serum and a 37 degrees C growth temperature. Time course analysis of this transition identified transcripts that are induced before germ tube initiation and shut off later in the developmental process. A strain deleted for the Efg1p and Cph1p transcription factors is defective in hyphae formation, and its response to serum and increased temperature is almost identical to the response of a wild-type strain grown at 37 degrees C in the absence of serum. Thus Efg1p and Cph1p are needed for the activation of the transcriptional program that is induced by the presence of serum.