A novel method for analysis of human T cell repertoires by real-time PCR.

T lymphocyte responses to challenges with multiple pathogens depend on the diversity of their T cell receptors (TcRs) that are heteroduplexes of alpha and beta chains. The regions of alpha and beta chains that define TcR specificity are encoded by rearranged variable (V) and joining (J) genes that are separated by variable numbers of nucleotides that encode the complementarity determining region 3 (CDR3). The assumption that a "healthy" T cell compartment exhibits broad TcR and CDR3 diversity has driven development of methods to evaluate diversity of TcR beta transcripts expressed by T lymphocyte populations and subpopulations in inflammatory sites and human malignancies. To that end, we have developed the BV:BJ matrix assay that uniquely generates a single statistic that describes TcR repertoire diversity and improves identification of beta transcripts expressed by expanded T cell clonotypes. The BV:BJ matrix uses rigorously selected primers specific for individual V and J genes to amplify beta transcripts in real-time PCRs driven by 533 BV:BJ primer pairs. The quantitative control of real-time PCRs produces Shannon entropy estimates of diversity that are reproducible over a range of template amounts and amenable to statistical analyses that have been difficult to perform with existing methods of repertoire analysis.

MHC class II epitope nesting modulates dendritic cell function and improves generation of antigen-specific CD4 helper T cells.

CD4 Th cells are critical to the development of coordinated immune responses to infections and tumors. Th cells are activated through interactions of the TCR with MHC class II complexed with peptide. T cell activation is dependent on the density of MHC peptide complexes as well as the duration of interaction of the TCR with APCs. In this study, we sought to determine whether MHC class II peptides could be modified with amino acid sequences that facilitated uptake and presentation with the goal of improving Th cell activation in vitro and in vivo. A model epitope derived from the murine folate receptor α, a self- and tumor Ag, was modified at its carboxyl terminus with the invariant chain-derived Ii-Key peptide and at its N terminus with a peptide that enhances uptake of Ag by APC. Modification of a peptide resulted in enhanced generation of high-avidity murine folate receptor α T cells that persisted in vivo and homed to sites of Ag deposition. The nesting approach was epitope and species independent and specifically excluded expansion of CD4 regulatory T cells. The resulting Th cells were therapeutic, enhanced in vivo helper activity and had an increased ability to resist tolerizing immune microenvironments. In addition to improved immunoadjuvants, this epitope modification strategy may be useful for enhancing ex vivo and in vivo generation of Th cells for preventing and treating diseases.

The application of real-time PCR to the analysis of T cell repertoires.

The diversity of T-cell populations is determined by the spectrum of antigen-specific T-cell receptors (TCRs) that are heterodimers of alpha and beta subunits encoded by rearranged combinations of variable (AV and BV), joining (AJ and BJ), and constant region genes (AC and BC). We have developed a novel approach for analysis of beta transcript diversity in mice with a real-time PCR-based method that uses a matrix of BV- and BJ-specific primers to amplify 240 distinct BV-BJ combinations. Defined endpoints (Ct values) and dissociation curves are generated for each BV-BJ combination and the Ct values are consolidated in a matrix that characterizes the beta transcript diversity of each RNA sample. Relative diversities of BV-BJ combinations in individual RNA samples are further described by estimates of scaled entropy. A skin allograft system was used to demonstrate that dissection of repertoires into 240 BV-BJ combinations increases efficiency of identifying and sequencing beta transcripts that are overrepresented at inflammatory sites. These BV-BJ matrices should generate greater investigation in laboratory and clinical settings due to increased throughput, resolution and identification of overrepresented TCR transcripts.

Distributions of single nucleotide polymorphisms in differential chromosome segments of congenic resistant strains that define minor histocompatibility antigens.

Minor histocompatibility antigens (MiHAs) stimulate the rejection of allografts when donors and recipients are matched at the major histocompatibility complex (MHC). The majority of identified autosomal MiHAs were generated by non-synonymous (NS) substitutions that alter MHC class I-binding peptides. The mosaic distribution of single nucleotide polymorphisms (SNPs) that distinguish inbred mouse strains led us to hypothesize that MiHA genes defined by congenic strains on C57BL/6 and C57BL/10 backgrounds map to chromosomal regions with relatively high numbers of NS SNPs that distinguish C57 strains from other common inbred strains. To test this hypothesis, we mapped the ends of differential chromosome segments of congenic strains, which define 12 MiHAs, relative to microsatellites and SNPs. The lengths of differential segments ranged from 9.7 to 105.9 Mbp in congenic strains where no attempts were made to select recombinants within these segments. There was no apparent correlation between differential segment length and number of backcrosses, suggesting that factors other than the number of opportunities for recombination affected the differential segment lengths in these congenics. These differential segments included higher numbers of NS SNPs that distinguish C57BL/6J from A/J, DBA/2J, and 129S1/J than would be predicted if these SNPs were uniformly distributed along the chromosomes. The most extreme case was the H8 congenic that included 74% of the SNPs on chromosome 14 within its 9.7-11.1 Mbp differential segment. These results point toward a direct relationship between the level of genomic divergence, as indicated by numbers of NS SNPs, and numbers of MiHAs that collectively determine the magnitude of allograft rejection.

Repertoires of T cell receptors expressed by graft-infiltrating T cells evolve during long-term recall responses to single minor histocompatibility antigens.

Long-lived mammals such as humans respond over decades with CTL and helper T lymphocytes to acute and chronic infections. Maintaining these extended recall responses requires established memory populations of sufficient size and diversity to effectively respond to these infections. Studies in mice have indicated that cytotoxic T cells that respond to secondary viral infections are principally those that were activated in primary responses and maintained through memory. However, long-term recall responses in humans must involve many more responses with increased opportunities for recruitment of naive T cells and competition between memory and naive cells. We hypothesized that increased numbers of antigenic challenges prolong selection pressure on responding T cells resulting in continuously changing populations characterized by evolving TCR repertoires. This hypothesis was tested by transplanting recipients with 10 sets of H4-incompatible skin allografts that were harvested at times of rejection for spectratyping of TCR alpha and beta transcripts expressed by graft-infiltrating T cells. Amplicons with single complementarity-determining region 3 (CDR3) lengths were sequenced, and CDR3-specific primers were used for amplifications to identify graft sets in which these sequences were present. The results showed that TCR repertoires did not stabilize in these extended recall responses since the majority of identified alpha and beta CDR3s was present in varying numbers of graft sets with only a minority being present throughout. Selection for specific beta CDR3s was suggested by observed associations between the extent of the recall responses and shortened CDR3 lengths and restricted distributions of net charges in CDR3s.

Expression of mRNA for a newly identified Pax5 exon is reduced in multiple myeloma.

Pax5 is a transcription factor that is critical in the bone marrow for differentiation and proliferation of B cells until the plasma cell stage. In Pax5(-/-) mice, B-cell development stalls at the pro-B-cell stage. Messenger RNA profiles of alternatively spliced isoforms of Pax5 in bone marrow frequently differ between multiple myeloma (MM) patients and healthy donors. We sought to determine if Pax5 mRNA profiles also differed in blood and unexpectedly detected the presence of a previously unreported exon that alters the amino acid code for the transactivating domain of Pax5 in CD138(-) B cells. This unique exon escapes detection by conventional analyses of RT-PCR products and may serve as a prototype for other exons, in other genes, that escape RT-PCR detection. Eight percent of tested human subjects were heterozygous for an allele with a nonsynonymous nucleotide substitution in the new exon, and one MM patient was homozygotic for this base difference. Subsequent analysis of plasma and B-cell populations from bone marrow revealed a markedly reduced mRNA expression of the new isoform in cells from MM patients when compared to cells from normal subjects.

Cysteine-tailed class I-binding peptides bind to CpG adjuvant and enhance primary CTL responses.

Immunostimulatory CpG motifs in synthetic oligonucleotides can be effective adjuvants for the priming of CTLs. We first observed that a single male-specific peptide (KCSRNRQYL) (HY2) was more efficient than another male-specific peptide (WMHHNMDLI) (HY1) at priming IFN-gamma-secreting CTLs in vivo when combined with lipid A and CpG and that it also visibly precipitated CpG. The addition of the six N-terminal residues (KCSRNR) from HY2 to HY1 yielded a peptide, KCSRNR-HY1, that both precipitated CpG and primed increased numbers of HY1-specific CTLs. We refer to this type of peptide as a primotope that includes a class I binding peptide tailed with amino acids that increase priming. Ala residues were substituted for the Arg/Lys residues (ACSANA-HY1), and these substitutions did not reduce in vivo priming potential. However, the substitution of Ala for Cys (KASRNR-HY1) resulted in the complete loss of priming, demonstrating the importance of Cys for in vivo priming when mixed with CpG. This result suggested that increased priming was based in disulfide bonding between Cys residues and internal phosphorothioate groups of synthetic CpG. The addition of Cys-bearing primotopes to radiolabeled CpG with a single thioate group resulted in the appearance of a new band that was inhibited by 1) Cys > Ala substitution and 2) reduction and alkylation of CpG. These results reveal a novel mechanism for complexing class I binding peptides and CpG adjuvant for development of new peptide-adjuvant combinations for vaccines for cancer and infectious diseases.

Development and immunophenotyping of squamous cell carcinoma xenografts: tools for translational immunology.

OBJECTIVES/HYPOTHESIS: The objectives of this study were to delineate methods for the development of primary squamous cell carcinoma (SCCHN) xenografts and to define human leukocyte antigen (HLA), melanoma-associated antigen (MAGE)-A3, and human papilloma virus (HPV) 16 antigenic expression in resultant cellular products. STUDY DESIGN: Prospective experimental model. METHODS: Freshly isolated SCCHN xenografts were established in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice using a variety of methods. Resultant tumors were analyzed for expression patterns of HLA-A, MAGE-A3, and HPV 16. Appropriate controls were included to ensure the presence of human RNA. RESULTS: Three xenografts were successfully established and passaged in vivo. Characterization of the resultant products revealed that one was positive for HLA-A2 at both the DNA and protein levels. One of the tumor lines expressed MAGE-A3, whereas none expressed HPV 16. CONCLUSIONS: Freshly isolated SCCHN can be used to generate primary xenografts. Characterization of select patterns of protein expression in established xenografts is a precursor to the development of a mouse model for SCCHN using tumor bearing animals reconstituted with autologous patient leukocytes.

Long-term bovine hematopoietic engraftment with clone-derived stem cells.

Therapeutic cloning by somatic cell nuclear transfer offers potential for treatment of a wide range of degenerative disease. Nuclear transplantation with neo (r)-marked somatic nuclei from 10-13-year-old cows was used to generate cloned bovine fetuses. Clone fetal liver (FL) hematopoietic stem cells (HSC) were transplanted into two busulfan-treated and one untreated nuclear donor cows. Hematopoiesis was monitored over 13-16 months by in vitro progenitor and HSC assays. Chimerism was demonstrated by PCR in blood, marrow, lymph nodes, and endothelium, peaking at levels of 9-17% in blood granulocytes but at lower levels in lymphocyte subsets (0.1-0.01%). Circulating progenitors showed high levels of chimerism (up to 60% neo (r+)) with persisting fetal features. At sacrifice, the animal that had no pre-transplant myelosupression showed persisting donor cells in blood and lymph nodes, and in marrow 0.25% of progenitor cells and a detectable fraction of stem cells were neo (r+). The fetal HSC showed a 10-fold competition advantage over adult HSC. Cloning generated histocompatible HSC capable of long-term multilineage engraftment in a large animal model.

Mitosis increases levels of secretory leukocyte protease inhibitor in keratinocytes.

Chronic wounds are a major health care burden. Multiple factors produced by healing wounds play important roles in efficient and orderly wound healing. Secretory leukocyte protease inhibitor (SLPI) is constitutively expressed in epithelial cells, and its expression is increased by inflammation. SLPI has antimicrobial activities and improves wound healing. We hypothesized that SLPI expression correlates with keratinocyte growth rate and is increased by epidermal growth factor (EGF). Keratinocytes were isolated from neonatal foreskin. Subconfluent and confluent culture conditions were used. SLPI-specific primers were designed for use in quantitative-competitive reverse-transcription polymerase chain reaction assays to detect and quantify SLPI mRNA expression levels. SLPI levels were increased 2-fold in subconfluent cultures and 1.6-fold in confluent cultures to which EGF had been added. Confluent cultures also demonstrated 7-fold (growth factor depleted) and 6-fold (EGF) increase in SLPI RNA copy number, compared to subconfluent cultures. The results indicate that EGF increases SLPI expression.

Altered mRNA expression of Pax5 and Blimp-1 in B cells in multiple myeloma.

Multiple myeloma (MM) is a plasma cell disorder that potentially initiates during an early stage of B-cell development. We encountered an unidentified isoform of B cell-specific activator protein (BSAP, or Pax5) in MM cells while performing differential analyses to compare mRNA expression in malignant and normal plasma cells. Pax5 is a transcription factor that plays a central role throughout B-cell development until the point of terminal differentiation. Our finding of this unique isoform prompted us to investigate Pax5 isoform usage in plasma cells and B-cell populations in other MM and healthy subjects. In contrast to normal Pax5 expression, we observed multiple isoforms of Pax5 in conjunction with low levels of expression of the full-length Pax5 in B cells from MM patients. The expressed isoforms in MM varied considerably from patient to patient, with no clear pattern. We also performed semiquantitative analyses of the mRNA expression levels of B lymphocyte-induced maturation protein (Blimp-1), because expression levels of Pax5 and Blimp-1 have been shown to be inversely correlated. We observed the expression of Blimp-1 in the B-cell populations in all 11 MM patients but in none of 11 healthy subjects. We hypothesize that premature Blimp-1 expression coupled to altered and deficient Pax5 expression causes some proliferating B cells to prematurely differentiate to plasma cells in MM.

Generation of histocompatible tissues using nuclear transplantation.

Nuclear transplantation (therapeutic cloning) could theoretically provide a limitless source of cells for regenerative therapy. Although the cloned cells would carry the nuclear genome of the patient, the presence of mitochondria inherited from the recipient oocyte raises questions about the histocompatibility of the resulting cells. In this study, we created bioengineered tissues from cardiac, skeletal muscle, and renal cells cloned from adult bovine fibroblasts. Long-term viability was demonstrated after transplantation of the grafts into the nuclear donor animals. Reverse transcription-PCR (RT-PCR) and western blot analysis confirmed that the cloned tissues expressed tissue-specific mRNA and proteins while expressing a different mitochondrial DNA (mtDNA) haplotype. In addition to creating skeletal muscle and cardiac "patches", nuclear transplantation was used to generate functioning renal units that produced urinelike fluid and demonstrated unidirectional secretion and concentration of urea nitrogen and creatinine. Examination of the explanted renal devices revealed formation of organized glomeruli- and tubule-like structures. Delayed-type hypersensitivity (DTH) testing in vivo and Elispot analysis in vitro suggested that there was no rejection response to the cloned renal cells. The ability to generate histocompatible cells using cloning techniques addresses one of the major challenges in transplantation medicine.