ImmunoPET of murine T cell reconstitution post-adoptive stem cell transplant using anti-CD4 and anti-CD8 cys-diabodies

Abstract

The proliferation and trafficking of T lymphocytes in immune responses are crucial events in determining inflammatory responses. To study whole body T lymphocyte dynamics non-invasively in vivo, we have generated anti-CD4 and -CD8 cys-diabodies (cDbs) derived from the parental antibody hybridomas GK1.5 and 2.43, respectively, for ⁸⁹Zr-immunoPET detection of helper and cytotoxic T cell populations.

Methods

Anti-CD4 and -CD8 cys-diabodies were engineered, produced via mammalian expression, purified using immobilized metal affinity chromatography, and characterized for T cell binding. The cys-diabodies were site-specifically conjugated to maleimide-desferrioxamine for ⁸⁹Zr radiolabeling and subsequent microPET/CT acquisition and ex vivo biodistribution in both wild type mice and a model of hematopoietic stem cell (HSC) transplantation.

Results

ImmunoPET and biodistribution studies demonstrate targeting and visualization of CD4 and CD8 T cell populations in vivo in the spleen and lymph nodes of wild type mice, with specificity confirmed through in vivo blocking and depletion studies. Subsequently, a murine model of HSC transplantation demonstrated successful in vivo detection of T cell repopulation at 2, 4, and 8 weeks post-HSC transplant using the ⁸⁹Zr-radiolabeled anti-CD4 and -CD8 cDbs.

Conclusion

These newly developed anti-CD4 and -CD8 immunoPET reagents represent a powerful resource to monitor T cell expansion, localization and novel engraftment protocols. Future potential applications of T cell targeted immunoPET include monitoring immune cell subsets in response to immunotherapy, autoimmunity, and lymphoproliferative disorders, contributing overall to preclinical immune cell monitoring.

INTRODUCTION

The ability to noninvasively monitor immune cells, specifically T cells, in the fields of oncology, immunotherapy, autoimmunity, and infection is difficult due to the complex nature of heterogeneous lymphocyte localization, proliferation and migration. Lymphocyte monitoring during immunotherapy protocols, such as detection of circulating lymphocytes from whole blood or tumor infiltrating lymphocytes from tissue biopsy, does not provide the full range of dynamic and spatial information needed. With the expanding implementation of immunotherapies, such as adoptive T cell transfer, hematopoietic stem cell or progenitor cell transfer, small molecule and antibody-based immunotherapies, and combinations thereof, whole body immuno-positron emission tomography (immunoPET) targeting of immune cell subtypes can potentially provide spatial and temporal information that is impossible utilizing current methods.

ImmunoPET takes advantage of the exquisite specificity and affinity of antibodies or antibody fragments and the sensitivity of PET (1–3). Intact antibodies have been engineered into bivalent antibody fragments such as the cys-diabody (cDb; dimer of scFv; Figure 1A) or minibody (Mb; dimer of scFv-C_H3) to enhance immunoPET imaging characteristics, including rapid clearance for high target-to-background images at short times post-injection, avidity, engineered sites for site-specific conjugation, and lack of Fc effector functions, among others (4).

Figure 1. Anti-CD4 GK1.5 cDb characterization.

(A) Schematic of intact antibody and engineered cys-diabodies for site-specific conjugation of fluorescent or metal chelator moieties via thiol-specific chemistry. (B) SDS/PAGE gel (left) of purified GK1.5 cDb (Lane 1) and mal488 conjugated GK1.5 cDb (Lane 2) for fluorescent flow cytometry binding assays (L = molecular weight ladder). The ultraviolet image (right) of the same gel shows mal488 conjugated to GK1.5 cDb. (C) Size exclusion chromatography demonstrated the conjugation to mal488 did not disrupt the diabody conformation. Reference arrows indicate albumin (66 kDa) at 20.8 min, carbonic anhydrase (29 kDa) at 24.7 min, and cytochrome C (12.4 kDa) at 27.4 min. (D) Flow cytometry of single cell suspensions from the blood, thymus, spleen, and lymph nodes of C57BL/6 mice compares the binding of commercial anti-CD4-APC-Cy7 clone GK1.5 (left panel) and mal488-GK1.5 cDb (right panel). Ab = antibody; FITC = fluorescein isothiocyanate; PE = phycoerythrin.

Non-antibody based methods to detect lymphocytes using PET include direct cell labeling of cells ex vivo (5–7), reporter gene imaging of ex vivo genetically modified T cells (8), or the use of metabolic probes such as 2-deoxy-2-(¹⁸F)fluoro-D-glucose ([¹⁸F]-FDG), 3′deoxy-3′-(¹⁸F)fluorothymidine ([¹⁸F]-FLT), 1-(2′-deoxy-2′-(¹⁸F)fluoroarabinofuranosyl) cytosine ([¹⁸F]-FAC), and 2′-deoxy-2′-(¹⁸F)fluoro-9-β-arabinofuranosylguanine ([¹⁸F]F-AraG) (9–13). Direct cell labeling suffers from limitations of radionuclide half-life, probe dilution due to cell division, and potential toxic effects due to the radiosensitivity of lymphocytes. Reporter gene tracking of T cells allows for longitudinal tracking, repeat monitoring and signal amplification due to cell division, but it requires the transfection of cells with exogenous DNA and the development of non-immunogenic reporters for translation (14, 15). The use of radiolabeled metabolic probes does not require ex vivo manipulation of cells but these probes are either not specific for T cells (e.g., [¹⁸F]-FDG and [¹⁸F]-FLT) or they target proliferating T cells in secondary lymphoid organs and fail to detect tumor-infiltrating lymphocytes (e.g., [¹⁸F]-FAC).

Hematopoietic stem cell (HSC) therapy has become an attractive approach for the treatment of multiple malignancies (16). Currently many stem or progenitor cell therapies involving T cell receptor (TCR) or chimeric antigen receptor (CAR) targeting epitopes expressed on malignant cells are under development for clinical translation (17–20). Previous work utilizing PET to detect hematopoietic stem cell transfer and immune cell engraftment employs reporter genes to image total cell engraftment as opposed to lineage specific repopulation (14, 21). Here we report the development of anti-CD4 and -CD8 cDbs radiolabeled with ⁸⁹Zr for direct immunoPET detection of CD4⁺ and CD8⁺ T cells with the goal of detecting helper and cytotoxic lymphocyte repopulation after HSC therapy.

MATERIALS AND METHODS

C57BL/6, C57BL/6 SJL and AKR mice were obtained from the Jackson Laboratories and housed and maintained by the Department of Laboratory Animal Medicine at the University of California, Los Angeles. The UCLA Chancellor’s Animal Research Committee approved protocols for all animal studies.

Detailed information regarding construction of anti-CD4 and -CD8 diabodies, protein expression and purification, flow cytometry, immunoPET, biodistribution and data analysis can be found in the supplemental materials.

Maleimide-DFO conjugation

The cDbs at 1–2.2 mg/mL were then reduced with 20-fold molar excess of tris(2-carboxyethyl)phosphine (TCEP; Pierce) for 30 min at room temperature. A 20-fold molar excess of N-(3,11,14,22,25,33-hexaoxo-4,10,15,21,26,32-hexaaaza-10,21,32-trihydroxytetratriacontane)malemide (malDFO; Macrocyclics) was then added and allowed to react for 2 h at room temperature. Excess malDFO was removed using PD-10 desalting columns (GE Healthcare) that were pre-equilibrated with PBS. Eluted protein was concentrated with Amicon Ultra centrifugal filters (0.5 mL and 10 kDa MWCO; Milipore). Conjugation efficiency was measured qualitatively by size exclusion chromatography using a Superdex 75 column on an AKTA purifier and SDS/PAGE-Coomassie stain analysis.

⁸⁹Zr radiolabeling

⁸⁹Zr-oxalate was obtained from Washington University School of Medicine, Division of Radiological Sciences, St. Louis Missouri or 3D Imaging LLC. Upon arrival, ⁸⁹Zr-oxalate was diluted with 40% (v/v) 2 M Na₂CO₃ and allowed to incubate for 3 min. The activity was then diluted with 2.5× volume of 1 M HEPES (pH 7.0). Final pH was checked with pHydrion plastic indicator strips (Micro Essential Laboratory) to confirm a pH of 7. MalDFO-conjugated protein was incubated for 1 h at room temperature at about 4–5 μCi/μg. Radiolabeling efficiency was measured by ITLC (Biodex Medical Systems) using 20 mM citrate buffer pH 5.6 as the mobile phase using a Wizard 3″ 1480 Automatic Gamma Counter (Perkin-Elmer). Protein was purified using either BioRad6 Spin columns or PD-10 columns equilibrated with PBS. Radiochemical purity was assessed by ITLC as above.

Hematopoietic Stem Cell Transplant

150 mg/kg 5-fluorouracil (APP Pharmaceuticals) was injected i.v. into six- to ten-week old C57BL/6 SJL mice as previously described (14). Five days post-treatment, the bone marrow was harvested and cultured overnight with the following growth factors: stem cell factor (SCF; 100 ng/mL), IL-3 (6 ng/mL), IL-6 (10 ng/mL; all from Peprotech), and 5% (vol/vol) conditioned media from WeHi-3 cells (ATCC). 5 × 10⁵ HSCs were injected i.v. into eight week-old C57BL/6 mice that had been lethally irradiated (900 rad) on the same day.

RESULTS

Anti-CD4 GK1.5 Cys-Diabody Characterization

The anti-CD4 GK1.5 cDb was successfully engineered, produced, and purified with a final purified yield of 0.36 mg/L. The purity and molecular weight of the cDb were confirmed by SDS-PAGE and size exclusion chromatography (SEC; Figure 1B&C). Following mild reduction, GK1.5 cDb was conjugated to Alexa Fluor 488 C5-maleimide (mal488), with a recovery of 61% after purification. The conjugation resulted in a dye-to-protein molar ratio of 1.5:1 and the mal488 was bound to the monomeric protein as seen by SDS/PAGE analysis and fluorescent imaging of the gel (Figure 1B). Importantly, the elution profiles of the GK1.5 cDb and the mal488-GK1.5 cDb on SEC were similar (~55 kDa), demonstrating the conjugation to the Cys-tag has not disrupted the diabody conformation (Figure 1C). Mal488-GK1.5 cDb binds to primary cells isolated from the peripheral blood, thymus, spleen and lymph nodes of C57BL/6 (BL/6) mice similarly to the commercial GK1.5-APC-Cy7 antibody (Figure 1D).

Anti-CD4 GK1.5 Cys-Diabody Targets CD4⁺ T Cells In Vivo

Conjugation of the GK1.5 cDb to maleimide-DFO (malDFO) did not alter the size exclusion profile of the DFO conjugated protein (Figure S1). The malDFO-GK1.5 cDb was radiolabeled with ⁸⁹Zr with a radiolabeling efficiency of 96.9 ± 1.5 % (range: 94 – 98%, n=5). After spin column purification, the radiochemical purity was >99.5% as shown by ITLC. The specific activity was 4.2 ± 1.6 μCi/μg (range: 1.6 – 5.5 μCi/μg, n=5).

Protein doses of 11.8 ± 1.4 μg (range: 10.3 – 14 μg, n=5) were injected intravenously via the tail vein into BL/6 mice. ImmunoPET acquisition at 4, 8 and 22 h post-injection showed a decrease in activity in the heart over time demonstrating blood clearance and radioactivity was detected with high contrast in various lymphoid organs and the kidney at 22 h post-injection (Figure 2A, top row). Targeting to the lymph nodes and spleen was confirmed by ex vivo biodistribution at 22 h post-injection (Figure 2B and Table S1). Interestingly, targeting to the thymus was not similar to other lymphoid organs.

Figure 2. Anti-CD4 small-animal PET of 89Zr-malDFO-GK1.5 cDb in wild-type, CD4-blocked, and CD4-depleted C57BL/6 mice.

(A) ⁸⁹Zr-malDFO-GK1.5 cDb was injected into wild-type (top), CD4-blocked (bolus, GK1.4 cDb [3 mg/kg]; middle), and CD4-depleted (3 consecutive intraperitoneal injections of GK1.5 Ab [25 mg/kg] over 3 d; bottom) C57BL/6 mice and imaged at 4, 8, and 22 h after injection. Images are represented as 25-mm maximum-intensity projections. (B) Ex vivo biodistribution at 22 h after injection of 89Zr-malDFO-GK1.5 cDb from wild-type, CD4-blocked, and CD4-depleted C57BL/6 mice. (C) Flow cytometry of single-cell suspensions from blood, thymus, spleen, and LNs of CD4-depleted C57BL/6 mice confirms depletion of CD4-expressing cells. Ab = antibody; ALN = axillary LN; B = bone; CLN = cervical LN; FITC = fluorescein isothiocyanate; ILN = inguinal LN; K = kidney; Li = liver; PLN = popliteal LN; Sp = spleen.

In order to evaluate specificity, the ⁸⁹Zr-malDFO-GK1.5 cDb was injected into BL/6 mice that received either a bolus co-injection of 3 mg/kg non-radiolabeled GK1.5 cDb or anti-CD4 antibody depletion treatment. ImmunoPET of CD4-blocked and CD4-depleted mice shows more rapid clearance from the blood at 4 h post-injection and lower uptake in lymphatic organs when compared to wild type BL/6 mice (Figure 2A). Ex vivo biodistribution of CD4-blocked and CD4-depleted BL/6 mice confirmed a significant decrease in the lymph nodes, spleen, and thymus when compared to wild type BL/6 mice (Figure 2B and Table S1). Taken together these results confirm specific targeting of ⁸⁹Zr-malDFO-GK1.5 cDb to lymphoid organs.

Anti-CD8 2.43 Cys-Diabody Characterization

The previously engineered anti-CD8 2.43 minibody (22) was successfully re-engineered as a cDb and purified at high purity (Figure S2) with a yield of 2.44 mg/L. Superdex 75 SEC confirmed the correct molecular weight of ~55 kDa (Figure S2).

The 2.43 cDb was conjugated to mal488 with a recovery of 64% after purification with a dye-to-protein molar ratio of 0.9:1. The mal488 was covalently coupled to the monomeric 2.43 cDb as seen by fluorescence detection on SDS/PAGE (Figure S2) and the SEC elution profiles of the unconjugated 2.43 cDb and the mal488-2.43 cDb were very similar, confirming successful conjugation to the C-terminal cysteine of the diabody (Figure S2). The mal488-2.43 cDb retains antigen specificity for CD8α Lyt2.2⁺ mouse strains (22) as demonstrated by flow cytometry analysis of primary cells isolated from the peripheral blood, thymus, spleen and lymph nodes (Figure 3).

Figure 3. Engineered anti-CD8 2.43 cDb retains antigen specificity.

Flow cytometry of single cell suspensions from the blood, thymus, spleen, and lymph nodes of Lyt2.2⁺ C57BL/6 (left panel) and Lyt2.1⁺ AKR mice (right panel) demonstrates that the mal488-2.43 cDb retains its antigen binding specificity.

Anti-CD8 2.43 Cys-Diabody Targets CD8⁺ T Cells In Vivo

The 2.43 cDb was conjugated to malDFO (Figure S2) and radiolabeled with ⁸⁹Zr with a radiolabeling efficiency of 85.5 ± 12.5% (range: 66 – 97%, n=6) and the radiochemical purity was >99.4% as shown by ITLC. The specific activity was 3.4 ± 0.9 μCi/μg (range: 2.5 – 4.6 μCi/μg, n=6).

Protein doses of 10.9 ± 0.6 μg (range: 10 – 11.6 μg, n=6) ⁸⁹Zr-malDFO-2.43 cDb were injected intravenously into either wild type BL/6 mice (Figure 4A, top panel) or BL/6 mice that received a bolus co-injection of 3 mg/kg non-radiolabeled 2.43 cDb (Figure 4B, bottom panel). ImmunoPET images of ⁸⁹Zr-malDFO-2.43 cDb in wild type BL/6 show high background at 4 h post-injection that clears by 20 h for high contrast images. Ex vivo biodistribution of ⁸⁹Zr-malDFO-2.43 cDb acquired at 22 h post-injection in wild type and CD8-blocked BL/6 mice shows significantly less uptake in the spleen and lymph nodes, demonstrating CD8 specificity in vivo (Figure 4B and Table S2). Targeting to the thymus of ⁸⁹Zr-malDFO-2.43 cDb was low and CD8-blocking does not decrease uptake.

Figure 4. Anti-CD8 microPET/CT of ⁸⁹Zr-malDFO-2.43 cDb in wild type and CD8-blocked C57BL/6 mice.

(A) ⁸⁹Zr-malDFO-2.43 cDb was injected into wild type (top panel) and CD8-blocked (bolus 3 mg/kg 2.43 cDb; bottom panel) C57BL/6 mice and imaged at 4, 8, and 22 h post-injection. Images are represented as coronal 25 mm MIPs. Abbreviations: CLN – cervical lymph node, ALN – axillary lymph node, Li – liver, Sp – Spleen, K – Kidney, ILN – inguinal lymph node, B – bone, and PLN – popliteal lymph node. (B) Transverse CT and PET/CT 0.5mm MIPs of the inguinal LNs (top panels) and spleen (bottom panels) in wt C57BL/6 mice at 22 h post-injection show PET coregistration with anatomical CT scans. Bars on coronal scan in top right panel of (A) indicate location of transverse images. (C) Ex vivo biodistribution at 22 h post-injection of ⁸⁹Zr-malDFO-2.43 cDb from wild type and CD8-blocked C57BL/6 mice.

T Cell Repopulation Post-Hematopoietic Stem Cell Transplant

BL/6 SJL (CD45.2⁺) HSCs were intravenously injected into lethally irradiated BL/6 (CD45.1⁺) mice to enable distinction between donor and host lymphocytes at two, four, and eight weeks post-HSC transplant using flow cytometry (Figure 5). At two weeks post-HSC transplant, the total donor cells were high in the peripheral blood and low in both the lymph nodes and spleen. By eight weeks, however, the donor cells are about 85% of the total CD45⁺ cells in the lymph nodes, spleen, and peripheral blood, demonstrating efficient HSC engraftment and T cell repopulation.

Figure 5. Flow cytometry analysis of CD4⁺ and CD8⁺ T cell reconstitution.

Total donor versus host cells (top row), donor versus host CD4⁺ T cells (middle row), and donor versus host CD8⁺ T cells (bottom row) at two weeks (left column), four weeks (middle column), and eight weeks (right column) post-HSC transplant (n = 3 or 4). Single cell suspensions from the lymph nodes, peripheral blood, and spleen of mice are stained with anti-CD45.1 (donor), anti-CD45.2 (host), anti-CD4, and anti-CD8 for flow cytometry analysis to quantify CD4⁺ and CD8⁺ T cell percentages.

When analyzing the CD4⁺ T cell engraftment (Figure 5, middle row), the total CD4 numbers at two weeks post-HSC transplant were very low in the peripheral blood and spleen (about 5 and 3%, respectively) but high in the LNs (~40%) with the majority of the CD4⁺ T cells being of host origin. At four weeks, the total CD4⁺ T cells in the peripheral blood and spleen increased to about 12% with the majority of the CD4⁺ T cells from donor origin. Interestingly, the total number of CD4⁺ T cells in the LNs at four weeks decreased (~28%), but the percentage of donor CD4⁺ T cells increased compared to the engraftment at two weeks post-HSC transplant. By eight weeks, the total CD4⁺ T cells in the lymph nodes, peripheral blood and spleen were ~35, 22, and 18%, respectively, with the majority of CD4⁺ T cells being of donor origin. Total CD8 cell numbers and repopulation of donor CD8⁺ T cells at two, four and eight weeks post-HSC transplant (Figure 5, bottom row) were similar to that of CD4, but the total numbers of CD8⁺ T cells were about half of the total CD4⁺ T cells. Thus, flow cytometry demonstrates effective repopulation of CD4⁺ and CD8⁺ T cells over time.

Anti-CD4 and -CD8 ImmunoPET Detection of T Cell Repopulation

⁸⁹Zr-malDFO-GK1.5 cDb and ⁸⁹Zr-malDFO-2.43 cDb were used to image CD4⁺ and CD8⁺ T cell reconstitution, respectively, in vivo at two, four and eight weeks post-HSC transplant (Figure 6 and Table S3). Detection of the lymph nodes from anti-CD4 and anti-CD8 immunoPET images in HSC transplanted mice increased from two to eight weeks post-HSC transplant. The biodistribution data of the anti-CD4 cDb, however, indicate that the %ID/g in the lymph nodes for the anti-CD4 repopulation was high at two and four weeks post-HSC transplant (axillary LNs: 75 ± 11 %ID/g and 79 ± 13 %ID/g, respectively) and decreased at eight weeks (39 ± 8.5 %ID/g). In the spleen, the %ID/g of ⁸⁹Zr-malDFO-GK1.5 cDb was moderate at two weeks post-HSC transplant (16 ± 2.0 %ID/g) and then increased at four and eight weeks post-HSC transplant (36 ± 3.4 %ID/g and 33 ± 3.1 %ID/g, respectively). The %ID/g for CD8 repopulation in both the spleen and the lymph nodes remained relatively constant from two to eight weeks post-HSC transplant (axillary LNs ranged from 18 – 20 %ID/g on average and spleen ranged from 17 – 28 %ID/g on average).

Figure 6. Anti-CD4 and -CD8 ⁸⁹Zr microPET/CT of T cell reconstitution post-HSC transplant.

(A) ⁸⁹Zr-malDFO-GK1.5 cDb and (B) ⁸⁹Zr-malDFO-2.43 cDb were injected into C57BL/6 mice at 2, 4, and 8 weeks post-HSC transplant. ImmunoPET images were acquired at 22 h post-injection. Bottom panels are ex vivo biodistributions at 22 h post-injection.

Representative computed tomography (CT) scans at two, four and eight weeks post-HSC transplant demonstrate a visible increase in size of the inguinal LN over time due to repopulation of the immune system (Figure S3). Inguinal LNs weighed 0.71 ± 0.12, 0.86 ± 0.20, and 2.0 ± 0.24 mg at two, four and eight weeks post-HSC transplant, respectively (Figure S3). These weights were used for estimating the total number of cells, the total CD4⁺ T cells, and total CD8⁺ T cells at two, four and eight weeks post-HSC transplant (Figure S3). This indicates that not only do the percentages of donor T cells change as seen by flow cytometry, but the total number of lymphocytes also increased during repopulation and this is visualized by both anti-CD4 and -CD8 immunoPET.

DISCUSSION

By engineering the GK1.5 and 2.43 parental antibodies into cys-diabodies, we have developed immunoPET fragments for the detection of helper CD4⁺ and cytotoxic CD8⁺ T cells in vivo. Importantly, we have demonstrated that both the GK1.5 and 2.43 cDbs retain a true cross-paired diabody conformation following conjugation via the engineered C-terminal cysteine. It is important to verify maintenance of the proper conformation of cDbs as different linker lengths have the potential to induce higher molecular weight multimers (23, 24) or convert into monovalent scFvs following reduction and site-specific conjugation (25). Flow cytometry was used to demonstrate that the GK1.5 and 2.43 cDbs retain the ability to bind primary CD4⁺ and CD8⁺ T cells, respectively. Furthermore, we used microPET in wild type immunocompetent BL/6 mice in combination with antigen depletion and/or antigen blocking to demonstrate either CD4 or CD8 specificity in vivo and demonstrated the ability to monitor T cell repopulation post-HSC transplant.

Minibodies (~80 kDa) and cys-diabodies (~50 kDa) have different routes of clearance due to the renal filtration cutoff of ~60 kDa. The use of the residualizing radiometals, such as ⁶⁴Cu and ⁸⁹Zr, conjugated to minibodies or cys-diabodies causes retention of radioactivity in the liver or kidney, respectively. Therefore, the high kidney uptake of both the ⁸⁹Zr-radiolabeled GK1.5 and 2.43 cys-diabodies is expected. The previously described ⁶⁴Cu-DOTA-2.43 minibody (22) suffered from aggregation problems leading to rapid clearance and high liver uptake but this allowed for high target-to-background images at only 4 hours post-injection. In the current work, the non-aggregating ⁸⁹Zr-malDFO-2.43 cDb demonstrated slower blood clearance than the respective 2.43 minibody and the improved time for imaging was 22 hours post-injection when the blood activity was 0.61 ± 0.06 %ID/g and inguinal LN:blood and spleen:blood ratios were 78:1 and 67:1, respectively. Previously, the 2.43 Mb derived from a CD8-depleting rat IgG2a antibody was shown to be non-depleting in vivo due to the lack of the complete Fc domain (22). The GK1.5 and 2.43 cDbs described in this work also lack the Fc domain and are expected to be non-depleting, which is critical in the development of immune cell targeting imaging agents.

An inherent problem of PET imaging of objects near or below the resolution of the PET scanner is the partial volume effect whereby the activity in a small organ is underestimated due to image reconstruction, attenuation, and positron path length prior to annihilation (26). Given the small animal scanner resolution of 1.4 mm FWHM (full width half maximal value), lymph nodes of about 1 mm in wild type mice, and the average positron path length of 1.1 mm for ⁸⁹Zr, a discrepancy exists between the %ID/g in lymph nodes obtained from PET imaging compared to the ex vivo biodistribution values. This discrepancy can be overcome by the use of partial volume correction to obtain accurate uptake values in small organs from PET images and our lab has derived recovery coefficients for both ⁸⁹Zr and ¹²⁴I using the Inveon microPET scanner (27). Partial volume correction of lymph nodes, especially when reduced in size at two weeks post-HSC transplant, will suffer from error due to the small volume regions of interest where the recovery coefficient changes greatly for small changes in volume and the detectability of the lymph nodes.

Although detection of activity in lymph nodes is underestimated using PET, we successfully detected CD4⁺ and CD8⁺ T cell reconstitution at two, four, and eight weeks post-HSC transplant using the ⁸⁹Zr-malDFO-GK1.5 and ⁸⁹Zr-malDFO-2.43 cys-diabodies. Detection at 2 weeks post-HSC transplant proved more difficult as the lymph node size is small post-irradiation. We were able to detect CD4⁺ T cells by immunoPET at 75 ± 11 %ID/g but not CD8⁺ T cells at 20 ± 0.60 %ID/g in lymph nodes of the same size (0.74 ± 0.12 mg), demonstrating that if enough activity accumulates in a small organ, it is detectable by PET. The partial volume effect is also evident over the eight weeks post-HSC transplant using the anti-CD8 ⁸⁹Zr-malDFO-2.43 cDb. The ex vivo biodistributions of ⁸⁹Zr-malDFO-2.43 cDb at two, four and eight weeks post-HSC transplant in the lymph nodes are consistently around 20–25 %ID/g, but the corresponding immunoPET images show an increase of signal in the lymph nodes from two to eight weeks post-HSC transplant. This increased detection is due to the increased size of the lymph nodes over time resulting from immune cell repopulation. It should be noted that PET images of the HSC transplant model were scaled to the same %ID/g for consistent representation.

Interestingly, the thymus showed low uptake in wild type mice using anti-CD4 and -CD8 cDbs when compared to other T cell-rich lymphoid organs such as the lymph nodes and the spleen, limiting their use as thymic immunoPET imaging agents. The parental anti-CD4 GK1.5 and anti-CD8 2.43 intact antibodies deplete CD4 or CD8 expressing cells in the thymus during antibody-based depletion therapy, so it is possible that the full Fc is required for efficient targeting to the thymus.

ImmunoPET and reporter genes provide different information in the context of TCR use in hematopoietic stem or progenitor cell therapy. Reporter genes allow for reporter probe uptake in the stem or progenitor cells primarily transduced and all successive cell types, such as T cells, B cells, monocytes, macrophages (14). Reporter genes provide an indication of total engraftment of donor cells but lack specificity for cell lineages. On the other hand, antibody-based immunoPET targeting lymphocytes provides an indication of CD4 or CD8 lymphocyte reconstitution, but cannot distinguish between host and donor lymphocytes. Imaging T cell expansion and localization, specifically tumor targeting, using lymphocyte-specific immunoPET could aid the monitoring of immunotherapeutic efficacy. In combination, reporter gene and CD4 and CD8 based immunoPET can provide information pertaining to the success of HSC transplant that was previously impossible.

CONCLUSION

Anti-CD4 and -CD8 immunoPET is a robust method for detection of lymphocytes in vivo and was shown to detect T cell repopulation in a model of syngeneic HSC transplantation. We believe immunoPET using radiolabeled antibody fragments specific for cell surface lineage markers will greatly aid the ability to monitor the dynamic nature of T cell subsets in models of disease and expand our knowledge of both T cells in vivo and immunotherapies.