Select sequencing of clonally expanded CD8+ T cells reveals limits to clonal expansion.

To permit the recognition of antigens, T cells generate a vast diversity of T cell receptor (TCR) sequences. Upon binding of the TCR to an antigen-MHC complex, T cells clonally expand to establish an immune response. To study antigen-specific T cell clonality, we have developed a method that allows selection of rare cells, based on RNA expression, before in-depth scRNA-seq (named SELECT-seq). We applied SELECT-seq to collect both TCR sequences and then transcriptomes from single cells of peripheral blood lymphocytes activated by a Mycobacterium tuberculosis (Mtb) lysate. TCR sequence analysis allowed us to preferentially select expanded conventional CD8+ T cells as well as invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells. The iNKT and MAIT cells have a highly similar transcriptional pattern, indicating that they carry out similar immunological functions and differ considerably from conventional CD8+ T cells. While there is no relationship between expression profiles and clonal expansion in iNKT or MAIT cells, highly expanded conventional CD8+ T cells down-regulate the interleukin 2 (IL-2) receptor alpha (IL2RA, or CD25) protein and show signs of senescence. This suggests inherent limits to clonal expansion that act to diversify the T cell response repertoire.

Isolation and characterization of NY-ESO-1-specific T cell receptors restricted on various MHC molecules.

Tumor-specific T cell receptor (TCR) gene transfer enables specific and potent immune targeting of tumor antigens. Due to the prevalence of the HLA-A2 MHC class I supertype in most human populations, the majority of TCR gene therapy trials targeting public antigens have employed HLA-A2-restricted TCRs, limiting this approach to those patients expressing this allele. For these patients, TCR gene therapy trials have resulted in both tantalizing successes and lethal adverse events, underscoring the need for careful selection of antigenic targets. Broad and safe application of public antigen-targeted TCR gene therapies will require (i) selecting public antigens that are highly tumor-specific and (ii) targeting multiple epitopes derived from these antigens by obtaining an assortment of TCRs restricted by multiple common MHC alleles. The canonical cancer-testis antigen, NY-ESO-1, is not expressed in normal tissues but is aberrantly expressed across a broad array of cancer types. It has also been targeted with A2-restricted TCR gene therapy without adverse events or notable side effects. To enable the targeting of NY-ESO-1 in a broader array of HLA haplotypes, we isolated TCRs specific for NY-ESO-1 epitopes presented by four MHC molecules: HLA-A2, -B07, -B18, and -C03. Using these TCRs, we pilot an approach to extend TCR gene therapies targeting NY-ESO-1 to patient populations beyond those expressing HLA-A2.

Long-term surviving cancer patients as a source of therapeutic TCR.

We have established a platform for the isolation of tumour-specific TCR from T cells of patients who experienced clinical benefit from cancer vaccination. In this review we will present the rationale behind this strategy and discuss the advantages of working with "natural" wild type TCRs. Indeed, the general trend in the field has been to use various modifications to enhance the affinity of such therapeutic TCRs. This was done to obtain stronger T cell responses, often at the cost of safety. We further describe antigen targets and recent in vitro and in vivo results obtained to validate them. We finally discuss the use of MHC class II-restricted TCR in immunotherapy. Typically cellular anti-tumour immune responses have been attributed to CD8 T cells; however, we isolated mainly CD4 T cells. Importantly, these MHC class II-restricted TCRs have the potential to induce broad, long lasting immune responses that enable cancer control. The use of CD4 T cell-derived TCRs for adoptive immunotherapy has so far been limited and we will here discuss their therapeutic potential.

A humanized TCR retaining authentic specificity and affinity conferred potent anti-tumour cytotoxicity.

The affinity of T-cell receptor (TCR) determines the efficacy of TCR-based immunotherapy. By using human leucocyte antigen (HLA)-A*02 transgenic mice, a TCR was generated previously specific for human tumour testis antigen peptide MAGE-A3112-120 (KVAELVHFL) HLA-A*02 complex. We developed an approach to humanize the murine TCR by replacing the mouse framework with sequences of folding optimized human TCR variable domains for retaining binding affinity. The resultant humanized TCR exhibited higher affinity and conferred better anti-tumour activity than its parent murine MAGE-A3 TCR (SRm1). In addition, the affinity of humanized TCR was enhanced further to achieve improved T-cell activation. Our studies demonstrated that the human TCR variable domain frameworks could provide support for complementarity-determining regions from a murine TCR, and retain the original binding activity. It could be used as a generic approach of TCR humanization.

Platypus TCRµ provides insight into the origins and evolution of a uniquely mammalian TCR locus.

TCRµ is an unconventional TCR that was first discovered in marsupials and appears to be absent from placental mammals and nonmammals. In this study, we show that TCRµ is also present in the duckbill platypus, an egg-laying monotreme, consistent with TCRµ being ancient and present in the last common ancestor of all extant mammals. As in marsupials, platypus TCRµ is expressed in a form containing double V domains. These V domains more closely resemble Ab V than that of conventional TCR. Platypus TCRµ differs from its marsupial homolog by requiring two rounds of somatic DNA recombination to assemble both V exons and has a genomic organization resembling the likely ancestral form of the receptor genes. These results demonstrate that the ancestors of placental mammals would have had TCRµ but it has been lost from this lineage.

Generating HPV specific T helper cells for the treatment of HPV induced malignancies using TCR gene transfer.

BACKGROUND: Infection with high risk Human Papilloma Virus (HPV) is associated with cancer of the cervix, vagina, penis, vulva, anus and some cases of head and neck carcinomas. The HPV derived oncoproteins E6 and E7 are constitutively expressed in tumor cells and therefore potential targets for T cell mediated adoptive immunotherapy. Effective immunotherapy is dependent on the presence of both CD4+ and CD8+ T cells. However, low precursor frequencies of HPV16 specific T cells in patients and healthy donors hampers routine isolation of these cells for adoptive transfer purposes. An alternative to generate HPV specific CD4+ and CD8+ T cells is TCR gene transfer. METHODS: HPV specific CD4+ T cells were generated using either a MHC class I or MHC class II restricted TCR (from clones A9 and 24.101 respectively) directed against HPV16 antigens. Functional analysis was performed by interferon-γ secretion, proliferation and cytokine production assays. RESULTS: Introduction of HPV16 specific TCRs into blood derived CD4+ recipient T cells resulted in recognition of the relevant HPV16 epitope as determined by IFN-γ secretion. Importantly, we also show recognition of the endogenously processed and HLA-DP1 presented HPV16E6 epitope by 24.101 TCR transgenic CD4+ T cells and recognition of the HLA-A2 presented HPV16E7 epitope by A9 TCR transgenic CD4+ T cells. CONCLUSION: Our data indicate that TCR transfer is feasible as an alternative strategy to generate human HPV16 specific CD4+ T helper cells for the treatment of patients suffering from cervical cancer and other HPV16 induced malignancies.

Transplantation of T-cell receptor α/ß-depleted allogeneic bone marrow in nonhuman primates.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a potentially curative treatment for hematologic cancers and chronic infections such as human immunodeficiency virus (HIV). Its success in these settings is attributed to the ability of engrafting immune cells to eliminate cancer cells or deplete the HIV reservoir (graft-versus-host effect [GvHE]). However, alloHSCT is commonly associated with graft-versus-host diseases (GvHDs) causing significant morbidity and mortality, thereby requiring development of novel allogeneic HSCT protocols and therapies promoting GvHE without GvHD using physiologically relevant preclinical models. Here we evaluated the outcomes of major histocompatibility complex-matched T-cell receptor α/ß-depleted alloHSCT in Mauritian cynomolgus macaques (MCMs). Following T-cell receptor α/ß depletion, bone marrow cells were transplanted into major histocompatibility complex-identical MCMs conditioned with total body irradiation. GvHD prophylaxis included sirolimus alone in two animals or tacrolimus with cyclophosphamide in another two animals. Posttransplant chimerism was determined by sequencing diagnostic single-nucleotide polymorphisms to quantify the amounts of donor and recipient cells present in blood. Animals treated posttransplant with sirolimus developed nearly complete chimerism with acute GvHD. In the cyclophosphamide and tacrolimus treatment group, animals developed mixed chimerism without GvHD, with long-term engraftment observed in one animal. None of the animals developed cytomegalovirus infection. These studies indicate the feasibility of alloHSCT engraftment without GvHD in an MHC-identical MCM model following complete myeloablative conditioning and anti-GvHD prophylaxis with posttransplant cyclophosphamide and tacrolimus. Further exploration of this model will provide a platform for elucidating the mechanisms of GvHD and GvHE and for testing novel alloHSCT modalities for HIV infection.

Competition-Based Cell Assay Employing Soluble T Cell Receptors to Assess MHC Class II Antigen Processing and Presentation.

Accurate assessment of antigen-specific immune responses is critical in the development of safe and efficacious biotherapeutics and vaccines. Endosomal processing of a protein antigen followed by presentation on major histocompatibility complex (MHC) class II constitute necessary steps in the induction of CD4+ T cell immune responses. Current preclinical methods for assessing immunogenicity risk consist of in vitro cell-based assays and computational prediction tools. Cell-based assays are time and labor-intensive while in silico methodologies have limitations. Here, we propose a novel cell-based assay capable of investigating an antigen's endosomal processing and MHC class II presentation capabilities. This novel assay relies on competition between epitopes for MHC class II binding and employs labeled soluble T cell receptors (sTCRs) as detectors of epitope presentation.

Two subsets of human T lymphocytes expressing gamma/delta antigen receptor are identifiable by monoclonal antibodies directed to two distinct molecular forms of the receptor.

Two mAbs directed to the TCR-gamma/delta were analyzed for their pattern of reactivity with CD3+WT31- cell populations or clones. In normal individuals, the BB3 mAb reacted with approximately 2/3 of peripheral blood CD3+WT31- lymphocytes, whereas delta-TCS-1 stained approximately 1/3 of such cells. In addition, the sum of the percentages of BB3+ and delta-TCS-1+ cells approximated the percentages of peripheral blood CD3+WT31- lymphocytes in seven normal donors tested. Also, in peripheral blood-derived polyclonal CD3+WT31- populations, cultured in IL-2, cells reacting with one or another mAb accounted for the whole cell population. On the other hand, only delta-TCS-1-reactive cells, but not BB3+ cells, could be detected in unfractionated as well as in CD4-8-thymocyte populations. Analysis of peripheral blood-derived CD3+WT31- clones showed that 70% of 72 clones analyzed reacted with BB3 mAb, but not with delta-TCS-1 mAb. On the other hand, delta-TCS-1 mAb stained the remaining BB3- clones. Five clones expressing medium-low amounts of CD8 antigen were BB3- delta-TCS-1+. Both types of clones lysed the Fc gamma receptor-bearing P815 target cell in the presence of anti-CD3 mAb (but not of mAb directed against HLA-DR, CD7 molecules, or TCR-alpha/beta). In this cytolytic assay, BB3 mAb induced target cell lysis only by BB3+ clones, whereas delta-TCS-1 mAb was effective only with delta-TCS-1+ clones. The CD3-associated surface molecules expressed by BB3+ or delta-TCS-1+ clones were analyzed after cell surface iodination and immunoprecipitation with the corresponding anti-TCR mAb or with anti-CD3 mAb (in digitonin-containing buffer). In SDS-PAGE, molecules immunoprecipitated from 13 BB3+ clones displayed, under nonreducing conditions, a molecular weight of 80 kD (in some cases, a minor 38-kD band could be detected). Under reducing conditions, two major components of 44 and 41 kD (and a minor component of 38 kD) were detected. On the other hand, TCR molecules immunoprecipitated from 11 different delta-TCS-1+ clones appeared as a diffuse band of 41-44 kD, both under reducing and nonreducing conditions (under non-reducing condition, an additional 38-kD band was present). Therefore, BB3+ cells express a disulphide-linked form of TCR-gamma/delta whereas delta-TCS-1+ cells express a non-disulphide-linked form.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of a third form of the human T cell receptor gamma/delta.

A subpopulation of the CD3+ peripheral T lymphocytes express the TCR-gamma/delta complex. Three distinct TCR-gamma forms that differ in size and in the ability to form a disulfide bridge with the TCR-delta subunit have been described. In this study we analyze the structural difference between the non-disulfide-linked 55-kD and 40-kD TCR-gamma chains. The 40-kD TCR-gamma form contains a smaller polypeptide backbone and carries less carbohydrate compared with the 55-kD TCR-gamma form. A cDNA clone corresponding to the 40-kD TCR-gamma subunit lacks one copy of the second exon of the constant region that is present in the other TCR-gamma subunit. This exon copy encodes part of the connector region that is located between the constant domain and the membrane spanning region. We show that the number of potential N-linked glycan attachment sites are the same for the two TCR-gamma forms. Since these attachment sites are located in the connector region we conclude that the connector region influences the amount of N-linked carbohydrates added to the core TCR-gamma polypeptide, probably by affecting the conformation of the protein. In contrast to the TCR-beta constant region usage, the TCR-gamma constant regions are unequally expressed. Virtually exclusive usage of disulfide-linked complexes were found in some individuals, while both the disulfide-linked and the 40-kD, non-disulfide-linked TCR-gamma forms were detected in other subjects. The ability to distinguish these TCR-gamma/delta forms now makes it possible to study the mechanisms that govern their selection and to determine if they correspond to functionally distinct isotypes.

A unique V-J-C-rearranged gene encodes a gamma protein expressed on the majority of CD3+ T cell receptor-alpha/beta- circulating lymphocytes.

We have recently described an mAb, anti-Ti gamma A, that recognizes an antigenic determinant carried by a TCR gamma chain. This antibody binds to approximately 3% of human PBLs and delineates a CD2+, CD3+, TCR-alpha/beta-, CD4-, CD8+/-, CD5+, NKH1-, and HLA class II- subset. The present study was designed to identify the gene encoding the Ti gamma A epitope. A first analysis was carried out on a previously characterized TCR gamma + fetal-cloned cell line termed F6C7. It was found that F6C7 cells have one gamma rearrangement on each chromosome: one joins V gamma 3 to J gamma 1, and the second joins V gamma 9 to J gamma P. Because only the latter allele appeared to be transcribed in the F6C7 lymphocytes, these data strongly suggested that anti-Ti gamma A mAb is specific for either a V gamma 9 or a V gamma 9-J gamma P-encoded peptide. To confirm this point, we studied an additional series of 13 randomly selected Ti gamma A+ cloned cells derived from peripheral blood of three distinct adult individuals. Each one of these lymphocytes was shown to both possess and transcribe a V gamma 9-J gamma P-C gamma 1-rearranged gene. It is therefore concluded that a predominant subpopulation of CD3+ TCR-alpha/beta- human circulating T lymphocytes (namely, the subset defined by anti-Ti gamma A mAb) surface expresses a gamma protein with a limited potential of variability from one cell to another.

Identification of shared antigenic determinants of the putative human T lymphocyte antigen receptor.

A mouse antiserum, anti-gp40,49 was obtained by immunizing BALB/c mice with the putative T cell antigen receptor isolated from HPB-MLT cells. This antiserum reacted with peripheral blood lymphocyte (PBL) and a panel of immunocompetent T cell lines and clones in each case precipitating from lysates of cells labeled by surface iodination, a disulfide-linked dimer consisting of an alpha subunit Mr (46,000-49,000) and a beta subunit Mr (40,000-45,000). Variability in Mr of the two subunits, particularly of the beta (light) subunit, was observed when the receptors of immunocompetent T cell lines with different antigen specificities were compared. Anti-gp40,49 serum reacted selectively with the alpha subunit after reduction and alkylation of the protein complex. These results confirm the relationship between the gp40,49 protein complex of HPB-MLT cells and the putative T cell antigen receptor on normal immunocompetent T cells and indicate that the alpha subunit of the human receptor expressed shared determinant(s) that are immunogenic in the mouse. Some features of the T cell antigen receptor appear to be unusual in that even with a xenoantiserum against the purified molecule, only antibodies against clonotypic determinants could be detected at the cell surface by quantitative immunofluorescence analysis.

The major histocompatibility complex-restricted antigen receptor on T cells. I. Isolation with a monoclonal antibody.

An antibody-secreting B cell hybridoma, KJ1-26.1, has been prepared from mice immunized with the T cell hybridoma DO-11.10, which recognizes chicken ovalbumin in association with I-Ad (cOVA/I-Ad). KJ1-26.1 blocks I-restricted antigen recognition by DO-11.10 and a subclone of this T cell hybridoma, DO-11.10.24, which has the same specificity for cOVA/I-Ad as its parent. KJ1-26.1 does not block I-restricted antigen recognition by any other T cell hybridoma tested, including a number of T cell hybridomas closely related to DO-11.10, with similar, but not identical, specificities for antigen/I. Moreover, KJ1-26.1 binds to DO-11.10 and DO-11.10.24, but not to any other T cell hybridomas tested, including three subclones of DO-11.10 that have lost the ability to recognize cOVA/I-Ad. Thus, in every regard KJ1-26.1 appears to be binding to all or part of the receptors for antigen/I on the T cell hybridoma DO-11.10. KJ1-26.1 appears to bind to approximately 15,000 molecules/cell on the surface of DO-11.10. The antibody precipitates an 80,000 dimer from the cells, which on reduction migrates as 40-44,000 monomers. The receptor(s) for antigen/I on DO-11.10 therefore includes molecules with these properties.

A large subpopulation of avian T cells express a homologue of the mammalian T gamma/delta receptor.

This report describes an avian TCR molecule, TCR1, whose molecular characteristics, signal-transducing property, and tissue distribution suggest that it is a homologue of the mammalian TCR-gamma/delta. TCR1+ cells are the first to be generated in the thymus during ontogeny, preceding other T3+ cells by approximately 3 d. Unlike their mammalian counterpart, TCR1+ cells constitute a relatively large subpopulation of peripheral T cells in mature chickens. These results suggest a phylogenetically important role for this receptor in T cell development and function.

T cell receptor-mediated negative selection of autoreactive T lymphocyte precursors occurs after commitment to the CD4 or CD8 lineages.

To identify the maturational stage(s) during which T cell receptor (TCR)-mediated positive and negative selection occurs, we followed the development of CD4+8- and CD4-8+ T cells from TCRlo CD4+8+ thymic blasts in the presence of different positive and negative selecting (major histocompatibility complex or Mls) elements. We describe novel lineage-committed transitional intermediates that are TCRmed CD4+8lo or TCRmed CD4lo8+, and that show evidence of having been positively selected. Furthermore, negative selection is not evident until after cells have attained one of the TCRmed transitional phenotypes. Accordingly, we propose that negative selection in normal mice occurs only after TCRlo CD4+8+ precursors have been positively selected into either the CD4 or CD8 lineage.

The antigen-specific, major histocompatibility complex-restricted receptor on T cells. VI. An antibody to a receptor allotype.

We have prepared a monoclonal antibody, KJ16-133, from the cells of a rat immunized with the purified receptor for antigen plus I-A of a BALB/c T cell hybridoma, DO-11.10. Unlike most other monoclonal anti-receptor antibodies that have been described before, KJ16-133 is not clone specific. It reacts with approximately 20% of the receptors on T cells of normal BALB/c mice. It also reacts with about the same percentage of antigen-specific, major histocompatibility complex (MHC)-restricted or allogeneic I-region specific T cell hybridomas. Reaction of KJ16-133 with a given T cell hybridoma does not seem to depend on the antigen specificity or MHC-restricting element of the T cell in question. The determinant recognized by KJ16-133 has some unexpected properties. It is absent in several strains of mice including SJL/J and SJA/20, but present on the T cells of most other commonly used strains. The determinant recognized therefore does not map to Igh. Our experiments suggest that a clone-specific "antiidiotypic" antibody and KJ16-133 recognize determinants on different parts of the receptor. For example, the binding of a clone-specific antibody to target T cells is relatively temperature insensitive, whereas KJ16-133 binds well to cells at 37 degrees C but poorly to cells at 4 degrees C. The determinant recognized by a clone-specific antibody is sensitive to reduction and alkylation of the receptor, whereas KJ16-133 reactivity is not. Finally, binding of KJ16-133 at saturating concentrations to target T cells does not block the binding of a clone-specific antibody. Similarly, binding of a clone-specific antibody only marginally inhibits binding of KJ16-133. Taken together, these results suggest that KJ16-133 is directed against an allelic determinant on T cells that may be close to the membrane, and not in the receptor binding site for antigen plus MHC. The antibody may recognize an allele of a constant region isotype, or an allele of a J region.

Identification of Igh-C-linked determinants on suppressor T cell hybrids and factors specific for L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT).

Hyperimmunization of BALB/c mice with concanavalin A-stimulated blasts from the Ig allotype-congenic strain, C.B20, results in the production of antibodies reactive with T cells in an allotype-restricted manner. Spleen cells from these hyperimmune BALB/c mice were used to generate a panel of hybridomas that secrete monoclonal antibodies, reactive, in an allotype-restricted manner, exclusively with T cells subpopulations, and in particular, reactive with suppressor T cell hybridomas and their secreted soluble factors. Two functional classes of antibodies were identified: those that react with single polypeptide-chain suppressor T cell factors (TsF1) and the suppressor T cell hybridomas that produce such factors, and those that react with two polypeptide-chain suppressor T cell factors (TsF2) and their corresponding suppressor T cell hybridomas. These two classes of antibody were used to isolate molecules from the membranes of the respective suppressor T cell hybrids that are functionally and structurally related to the secreted suppressor T cell factors, suggesting a receptor function for these molecules.

Lck regulates the tyrosine phosphorylation of the T cell receptor subunits and ZAP-70 in murine thymocytes.

The Src-family and Syk/ZAP-70 family of protein tyrosine kinases (PTK) are required for T cell receptor (TCR) functions. We provide evidence that the Src-family PTK Lck is responsible for regulating the constitutive tyrosine phosphorylation of the TCR zeta subunit in murine thymocytes. Moreover, ligation of the TCR expressed on thymocytes from Lck-deficient mice largely failed to induce the phosphorylation of TCR-zeta, CD3 epsilon, or ZAP-70. In contrast, we find that the TCR-zeta subunit is weakly constitutively tyrosine phosphorylated in peripheral T cells isolated from Lck-null mice. These data suggest that Lck has a functional role in regulation of TCR signal transduction in thymocytes. In peripheral T cells, other Src-family PTKs such as Fyn may partially compensate for the absence of Lck.

Periplasmic expression of soluble single chain T cell receptors is rescued by the chaperone FkpA.

BACKGROUND: Efficient expression systems exist for antibody (Ab) molecules, which allow for characterization of large numbers of individual Ab variants. In contrast, such expression systems have been lacking for soluble T cell receptors (TCRs). Attempts to generate bacterial systems have generally resulted in low yields and material which is prone to aggregation and proteolysis. Here we present an optimized periplasmic bacterial expression system for soluble single chain (sc) TCRs. RESULTS: The effect of 1) over-expression of the periplasmic chaperon FkpA, 2) culture conditions and 3) molecular design was investigated. Elevated levels of FkpA allowed periplasmic soluble scTCR expression, presumably by preventing premature aggregation and inclusion body formation. Periplasmic expression enables disulphide bond formation, which is a prerequisite for the scTCR to reach its correct fold. It also enables quick and easy recovery of correctly folded protein without the need for time-consuming downstream processing. Expression without IPTG induction further improved the periplasmic expression yield, while addition of sucrose to the growth medium showed little effect. Shaker flask yield of mg levels of active purified material was obtained. The Valphabeta domain orientation was far superior to the Vbetaalpha domain orientation regarding monomeric yield of functionally folded molecules. CONCLUSION: The general expression regime presented here allows for rapid production of soluble scTCRs and is applicable for 1) high yield recovery sufficient for biophysical characterization and 2) high throughput screening of such molecules following molecular engineering.

Feasibility of real-time in vivo 89Zr-DFO-labeled CAR T-cell trafficking using PET imaging.

INTRODUCTION: Chimeric antigen receptor (CAR) T-cells have been recently developed and are producing impressive outcomes in patients with hematologic malignancies. However, there is no standardized method for cell trafficking and in vivo CAR T-cell monitoring. We assessed the feasibility of real-time in vivo 89Zr-p-Isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS, DFO) labeled CAR T-cell trafficking using positron emission tomography (PET). RESULTS: The 89Zr-DFO radiolabeling efficiency of Jurkat/CAR and human peripheral blood mononuclear cells (hPBMC)/CAR T-cells was 70%-79%, and cell radiolabeling activity was 98.1-103.6 kBq/106 cells. Cell viability after radiolabeling was >95%. Cell proliferation was not significantly different during the early period after radiolabeling, compared with unlabeled cells; however, the proliferative capacity decreased over time (day 7 after labeling). IL-2 or IFN-γ secretion was not significantly different between unlabeled and labeled CAR T-cells. PET/magnetic resonance imaging in the xenograft model showed that most of the 89Zr-DFO-labeled Jurkat/CAR T-cells were distributed in the lung (24.4% ± 3.4%ID) and liver (22.9% ± 5.6%ID) by one hour after injection. The cells gradually migrated from the lung to the liver and spleen by day 1, and remained stable in these sites until day 7 (on day 7: lung 3.9% ± 0.3%ID, liver 36.4% ± 2.7%ID, spleen 1.4% ± 0.3%ID). No significant accumulation of labeled cells was identified in tumors. A similar pattern was observed in ex vivo biodistributions on day 7 (lung 3.0% ± 1.0%ID, liver 19.8% ± 2.2%ID, spleen 2.3% ± 1.7%ID). 89Zr-DFO-labeled hPBMC/CAR T-cells showed a similar distribution, compared with Jurkat/CAR T-cells, on serial PET images. CAR T cell distribution was cross-confirmed by flow cytometry, Alu polymerase chain reaction, and immunohistochemistry. CONCLUSION: Real-time in vivo cell trafficking is feasible using PET imaging of 89Zr-DFO-labeled CAR T-cells. This can be used to investigate cellular kinetics, initial in vivo biodistribution, and safety profiles in future CAR T-cell development.