Use of plasmid DNA for vaccination has been demonstrated quite successfully in small rodents. However, some of the many challenges of DNA vaccine development are the relatively low performance obtained in larger animals and a generally weak mucosal immune response. Vaccination through salivary gland (SG) cannulation and delivery of aqueous solutions of DNA is one potential solution. The scalability of SG DNA vaccination was tested in multiple animal models; antigen specific titers above 10,000 were demonstrated in dogs and rats. Immune responses were also present at a variety of mucosal sites. In conclusion, our data demonstrate that DNA vaccination to the SG presents a unique and advantageous method for eliciting systemic and mucosal immune responses.

Although the maturation and export of T cells from the thymus has been extensively studied, the movement of cells in the opposite direction has been less well documented. In particular, the question of whether T cells which have been activated by antigen in the periphery are more likely to return to the thymus had been raised but not clearly answered. We examined this issue by activating T cells present in the periphery with their cognate antigen, and assessing migration to the thymus. TCR-transgenic cells from OT-I mice (Thy1.2+), which recognise the ovalbumin peptide OVA257–264 in the context of H-2Kb, were transferred into otherwise unmanipulated Thy1.1+ C57BL/6 mice. Recipient mice were injected i.v. with 5 μg peptide (SIINFEKL) approximately 24 hours later. The numbers of donor-derived (Thy1.2+) cells in the thymus and peripheral lymphoid tissue were determined. The results clearly show increased numbers of transgenic cells in the thymus 3 days after antigenic stimulation. However, since numbers of transgenic cells increased in the spleen and LN in about the same proportion, the data do not support the notion that there is highly increased selective migration of activated T cells to the thymus. Rather, they suggest that a sample of peripheral cells enters the thymus each day, and that the mature immigrants detected in the thymus merely reflect the contents of the peripheral T cell pool.

Using a mouse bone marrow transplantation model, the authors evaluated a Moloney murine leukemia virus (MMLV)-based vector encoding 2 anti-human immunodeficiency virus genes for long-term expression in blood cells. The vector also encoded the human nerve growth factor receptor (NGFR) to serve as a cell-surface marker for in vivo tracking of transduced cells. NGFR(+) cells were detected in blood leukocytes of all mice (n=16; range 16%-45%) 4 to 5 weeks after transplantation and were repeatedly detected in blood erythrocytes, platelets, monocytes, granulocytes, T cells, and B cells of all mice for up to 8 months. Transgene expression in individual mice was not blocked in the various cell lineages of the peripheral blood and spleen, in several stages of T-cell maturation in the thymus, or in the Lin(-/lo)Sca-1(+) and c-kit(+)Sca-1(+) subsets of bone marrow cells highly enriched for long-term multilineage-reconstituting activity. Serial transplantation of purified NGFR(+)c-kit(+)Sca-1(+) bone marrow cells resulted in the reconstitution of multilineage hematopoiesis by donor type NGFR(+) cells in all engrafted mice. The authors concluded that MMLV-based vectors were capable of efficient and sustained transgene expression in multiple lineages of peripheral blood cells and hematopoietic organs and in hematopoietic stem cell (HSC) populations. Differentiation of engrafting HSC to peripheral blood cells is not necessarily associated with dramatic suppression of retroviral gene expression. In light of earlier studies showing that vector elements other than the long-terminal repeat enhancer, promoter, and primer binding site can have an impact on long-term transgene expression, these findings accentuate the importance of empirically testing retroviral vectors to determine lasting in vivo expression.

Ex vivo cell cycling of hematopoietic stem cells (HSC), a subset of primitive hematopoietic progenitors (PHP) with engrafting capacity, is required for transduction with retroviral vectors and to increase transplantable HSC numbers. However, induction of division of HSC ex vivo also may lead to differentiation and loss of in vivo marrow repopulating potential. We evaluated mobilized peripheral blood (MPB) PHP for maintenance of stem cell function after ex vivo culture under conditions that we show can induce cycling of a majority of PHP with minimal differentiation. The following methods were combined: cell labeling with the division tracking dye carboxyfluorescein-diacetate succinimidylester (CFSE), analysis of primitive cell surface marker expression, an ex vivo PHP assay, and an in vivo marrow repopulating assay. MPB-purified CD34+Thy-1+ cells were labeled with CFSE dye and cultured for 112 hours in serum-deprived medium in the presence of the cytokine combinations of thrombopoietin (TPO), flt3 ligand (FL), and c-kit ligand (KL), or TPO, FL, and interleukin 6 (IL-6). Both cytokine combinations supported division of greater than 95% of cells within 112 hours with an average 2.1-fold (TPO, FL, KL) or 1.3-fold (TPO, FL, IL-6) increase in total cell numbers. An average of 21.6% (TPO, FL, KL) and 27.4% (TPO, FL, IL-6) of the divided cells still expressed the Thy-1 marker after 112 hours. Functional assays were performed to compare cultured and uncultured cells. CD34+Thy-1+CFSElo (post division) cells showed maintenance of cobblestone area-forming cell (CAFC) frequency (a mean of 1/9.0) relative to the starting population of uncultured CD34+Thy-1+ cells (a mean of 1/8.4). In contrast, CD34+ cells that had lost Thy-1 expression during culture (CD34+Thy-1−CFSElo) showed a mean 5.8-fold reduction in CAFC frequency (a mean of 1/52.5). Only the Thy-1–expressing fraction of cells post culture could engraft in vivo in the SCID-hu bone assay. Because the majority of HSC functional activity post culture was found in the CD34+Thy-1+ fraction, we focused on this fraction for subsequent analysis. CFSE labeling allows segregation and purification by flow cytometry of cells having undergone discrete numbers of divisions during culture. Very few cells that divided more than four times in culture still expressed Thy-1. Cells that retained expression of Thy-1 during culture retained CAFC activity relative to fresh CD34+Thy-1+ cells, after undergoing at least two divisions. CAFC frequency decreased after four divisions in culture with TPO, FL, and KL or after three divisions in TPO, FL, and IL-6. We then compared populations of Thy-1+ cells that had undergone sequential numbers of divisions in culture for their ability to engraft in the SCID-hu bone assay. Engrafting ability was retained throughout four divisions in both cytokine combinations. These data demonstrate that primitive MPB CD34+ cells maintain HSC function coincident with Thy-1 expression while undergoing two to four divisions under these culture conditions. Essentially all CD34+ Thy-1+ cells divided under the conditions tested, promoting susceptibility to retroviral transduction.

T lymphocytes expressing both CD4 and CD8 are the predominant cell type in the thymic cortex but are extremely rare outside the thymus of normal mice. In this article, we show that if precursor thymocytes (CD4-CD8-) from fetal or adult donors are injected i.v. into irradiated recipients, some of these cells will lodge in lymph nodes and develop into both CD4+CD8+ (double-positive) and CD4+ or CD8+ (single-positive) cells. This phenomenon also occurred in thymectomized recipients, strongly suggesting it is genuine extrathymic development. Prethymic precursors (e.g., fetal liver), were unable to use the lymph node for T cell development, without thymic processing. The data suggest that given unusual circumstances (irradiation or thymectomy and availability of appropriate precursors), the lymph nodes can support T cell development.

BACKGROUNDIncreasing use of phenotypically-enriched stem cell populations for clinical hematopoietic transplants has led to an urgent demand for a reliable, rapid and simple functional assay which would provide an estimation of the reconstituting potential of cells prior to transplantation.METHODSWe have developed a 2-week quantitative, stroma-free assay to measure the frequency of primitive progenitors within hematopoietic cell samples. This relatively short-term assay provides frequency information which correlates with that measured by a 5-week stroma-dependent CAFC assay. Cells with the phenotype CD34+Thy-1+ were purified by fluorescence-activated cell sorting from peripheral blood apheresis products of multiple myeloma patients mobilized with cytoxan and GM-CSF. CD34+Thy-1+ cells were plated at limiting dilution into microtiter wells and cultured in an Iscove's based serum-deprived culture medium, supplemented with the cytokines, interleukin (IL)-3, IL-6, G-CSF, Flk2/Flt3 ligand (FL) and Kit ligand (KL). After 2 weeks, cell proliferation in individual wells was quantified by microscopy and bright-field imaging, or by using a fluorescent nucleic acid-binding dye and fluorimetry. Poisson statistics were used to calculate the frequency of wells containing cells with high proliferative potential (wells containing > or = 500 cells).RESULTSProgenitor cell frequencies generated using this assay were compared by linear regression analysis to those generated from 32 parallel CAFC and CFU-C assays performed on the same patient samples. Correlations were r = 0.80, r2 = 0.65, and r = 0.76, r2 = 0.58, respectively; these correlations were highly significant (p < 10(-7)).DISCUSSIONThis limiting dilution assay should more directly quantitate the potential of primitive hematopoietic cells than a CFU-C assay. It also has advantages over both the CAFC and the CFU-C assay, in that scoring has been automated, making it simple, rapid, and objective compared with manual cobblestone area or colony counting. The described limiting dilution assay may provide a useful alternative to assays currently used to evaluate the viability and proliferative potential of purified hematopoietic cells intended for transplant.

NK1.1(+)alpha betaTCR(+) (NKT) cells have several important roles including tumor rejection and prevention of autoimmune disease. Although both CD4(+) and CD4(-)CD8(-) double-negative (DN) subsets of NKT cells have been identified, they are usually described as one population. Here, we show that NKT cells are phenotypically, functionally and developmentally heterogeneous, and that three distinct subsets (CD4(+), DN and CD8(+)) are differentially distributed in a tissue-specific fashion. CD8(+) NKT cells are present in all tissues but the thymus, and are highly enriched for CD8alpha(+)beta(-) cells. These subsets differ in their expression of a range of cell surface molecules (Vbeta8, DX5, CD69, CD45RB, Ly6C) and in their ability to produce IL-4 and IFN-gamma, with splenic NKT cell subsets producing lower levels than thymic NKT cells. Developmentally, most CD4(+) and DN NKT cells are thymus dependent, in contrast to CD8(+) NKT cells, and are also present amongst recent thymic emigrants in spleen and liver. TCR Jalpha281-deficient mice show a dramatic deficiency in thymic NKT cells, whereas a significant NKT cell population (enriched for the DN and CD8(+) subsets) is still present in the periphery. Taken together, this study reveals a far greater level of complexity within the NKT cell population than previously recognized.

Various combinations of cytokines have profoundly different effects on inhibition of apoptosis and stimulation of self-renewal division of hematopoietic stem cells (HSC) in short-term, ex vivo culture. Our goal was to quantitate expansion of cells with a primitive CD34+ Thy-1+ phenotype, as well as cell cycling, division history, differentiation, and apoptosis of CD34+ cells enriched from normal donor mobilized peripheral blood (MPB) cells. The balance of these parameters determines the net number of transplantable HSC produced in ex vivo cultures. Comparing several different combinations of cytokines added to 90-hour cultures of MPB CD34 cells, thrombopoietin (TPO), flt3 ligand (FL), and c-kit ligand (KL) gave the best result, with the lowest percentage of apoptotic cells and a mean 1.2-fold increase in the number of CD34+ Thy-1+ cells. A combination of interleukin 3 (IL-3), interleukin 6 (IL-6), and leukemia inhibitory factor (LIF) gave the worst outcome, including a decrease of CD34+ Thy-1+ cell number to a mean of 30% of the starting cell number. Cell division history was tracked using the dye 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE). Division of CD34+ Thy-1+ cells was faster and more synchronous in TPO, FL, and KL than in IL-3, IL-6, and LIF, which left a significant proportion of CD34+ cells undivided. Such detailed analyses of short-term, ex vivo cultures generated "replication scores," which allowed prediction of a sixfold improvement of the efficiency of gene transduction of primitive hematopoietic progenitors from MPB, using TPO, FL, and KL to replace IL-3, IL-6, and LIF. Analysis of retroviral transduction efficiency confirmed the increase of transgene expression from MPB primitive hematopoietic progenitors assayed after stromal culture was fivefold, validating the usefulness of multiparameter analysis of short-term cultures for survival and replication of CD34+ Thy-1+ cells.

Inefficient retroviral-mediated gene transfer to human hematopoietic stem cells (HSC) and insufficient gene expression in progeny cells derived from transduced HSC are two major problems associated with HSC-based gene therapy. In this study we evaluated the ability of a murine stem cell virus (MSCV)-based retroviral vector carrying the low-affinity human nerve growth factor receptor (NGFR) gene as reporter to maintain gene expression in transduced human hematopoietic cells. CD34(+) cells lacking lineage differentiation markers (CD34(+)Lin-) isolated from human bone marrow and mobilized peripheral blood were transduced using an optimized clinically applicable protocol. Under the conditions used, greater than 75% of the CD34(+) cell population retained the Lin- phenotype after 4 days in culture and at least 30% of these expressed a high level of NGFR (NGFR+) as assessed by fluorescence-activated cell sorter analysis. When these CD34(+)Lin-NGFR+ cells sorted 2 days posttransduction were assayed in vitro in clonogenic and long-term stromal cultures, sustained reporter expression was observed in differentiated erythroid and myeloid cells derived from transduced progenitors, and in differentiated B-lineage cells after 6 weeks. Moreover, when these transduced CD34(+)Lin-NGFR+ cells were used to repopulate human bone grafts implanted in severe combined immunodeficient mice, MSCV-directed NGFR expression could be detected on 37% +/- 6% (n = 5) of the donor-type human cells recovered 9 weeks postinjection. These findings suggest potential utility of the MSCV retroviral vector in the development of effective therapies involving gene-modified HSC.

Recent studies have opened the possibility that quiescent, G(0)/G(1) hematopoietic stem cells (HSC) can be gene transduced; lentiviruses (such as HIV type 1, HIV) encode proteins that permit transport of the viral genome into the nucleus of nondividing cells. We and others have recently demonstrated efficient transduction by using an HIV-1-based vector gene delivery system into various human cell types including human CD34(+) cells or terminally differentiated neurons. Here we compare the transduction efficiency of two vectors, HIV-based and murine leukemia virus (MuLV)-based vectors, on untreated and highly purified human HSC subsets that are virtually all in G(0)/G(1.) The HIV vector, but not MuLV vector supernatants, transduced freshly isolated G(0)/G(1) HSC from mobilized peripheral blood. Single-step transduction using replication-defective HIV resulted in HSC that expressed the green fluorescent protein (GFP) transgene while retaining their stem cell phenotype; clonal outgrowths of these GFP(+) HSC on bone marrow stromal cells fully retained GFP expression for at least 5 weeks. MuLV-based vectors did not transduce resting HSC, as measured by transgene expression, but did so readily when the HSC were actively cycling after culture in vitro for 3 days in a cytokine cocktail. These results suggest that resting HSC may be transduced by lentiviral-based, but not MuLV, vectors and maintain their primitive phenotype, pluripotentiality, and at least in vitro, transgene expression.

CD34(+)Thy-1(+)Lin- cells are enriched for primitive hematopoietic progenitor cells (PHP), as defined by the cobblestone area-forming cell (CAFC) assay, and for bone marrow (BM) repopulating hematopoietic stem cells (HSC), as defined by the in vivo SCID-hu bone assay. We evaluated the effects of different cytokine combinations on BM-derived PKH26-labeled CD34(+)Thy-1(+)Lin- cells in 6-day stroma-free cultures. Nearly all (>95%) of the CD34(+)Thy-1(+)Lin- cells divided by day 6 when cultured in thrombopoietin (TPO), c-kit ligand (KL), and flk2/flt3 ligand (FL). The resulting CD34(hi) PKHlo (postdivision) cell population retained a high CAFC frequency, a mean 3.2-fold increase of CAFC numbers, as well as a capacity for in vivo marrow repopulation similar to freshly isolated CD34(+)Thy-1(+)Lin- cells. Initial cell division of the majority of cells occurred between day 2 and day 4, with minimal loss of CD34 and Thy-1 expression. In contrast, cultures containing interleukin-3 (IL-3), IL-6, and leukemia inhibitory factor contained a mean of 75% of undivided cells at day 6. These CD34(hi) PKHhi cells retained a high frequency of CAFC, whereas the small population of CD34(hi) PKHlo postdivision cells contained a decreased frequency of CAFC. These data suggest that use of a combination of TPO, KL, and FL for short-term culture of CD34(+)Thy-1(+)Lin- cells increases the number of postdivision PHP, measured as CAFC, while preserving the capacity for in vivo engraftment.

The primary role of the thymus is to provide mature T cells for the peripheral immune system. The mechanisms involved in the cellular export processes are as yet unknown. In this study, we examined the ability of 2-acetyl-4-tetrahydroxybutylimidazole (THI), an agent widely used as a component of ammonia caramel food colouring, to inhibit T-cell export from the thymus. BALB/c mice were maintained on drinking water containing THI for 5 days. The mice showed a twofold increase in the total number of mature medullary thymocytes (CD4+CD8- and CD4-CD8+) as well as a slight decrease in the total number of immature double-positive cells (CD4+CD8+). The mature single-positive thymocytes were found to express high levels of the homing molecule L-selectin, suggesting that these potential emigrants were prevented from leaving the thymus. To confirm this, THI-treated mice were injected intrathymically with fluorescein isothiocyanate and the number of labelled T cells appearing in the lymph nodes and spleen was determined 16 hr later. A 10-fold decrease in the number of CD4+ and CD8+ recent thymic emigrants in the lymph nodes and spleen of THI-treated mice was observed. Previous studies have shown that THI does not affect other aspects of thymocyte development, such as proliferation and differentiation. Taken together, these results suggest that the immunosuppressive effects of THI may be due, in part, to preventing of the final step of T-cell export out of the thymus.

In humans, autologous transplants derived from bone marrow (BM) usually engraft more slowly than transplants derived from mobilized peripheral blood. Allogeneic BM transplants show a further delay in engraftment and have an apparent requirement for donor T cells to facilitate engraftment. In mice, Thy-1.1(lo)Lin-/loSca-1+ hematopoietic stem cells (HSCs) are the principal population in BM which is responsible for engraftment in syngeneic hosts at radioprotective doses, and higher doses of HSCs can radioprotect an allogeneic host in the absence of donor T cells. Using the mouse as a preclinical model, we wished to test to what extent engraftment kinetics was a function of HSC content, and whether at high doses of c-Kit+Thy-1.1(lo)Lin-/loSca-1+ (KTLS) cells rapid allogeneic engraftment could also be achieved. Here we demonstrate that engraftment kinetics varied greatly over the range of KTLS doses tested (100-10,000 cells), with the most rapid engraftment being obtained with a dose of 5,000 or more syngeneic cells. Mobilized splenic KTLS cells and the rhodamine 123(lo) subset of KTLS cells were also able to engraft rapidly. Higher doses of allogeneic cells were needed to produce equivalent engraftment kinetics. This suggests that in mice even fully allogeneic barriers can be traversed with high doses of HSCs, and that in humans it may be possible to obtain rapid engraftment in an allogeneic context with clinically achievable doses of purified HSCs.

638 Previous studies have demonstrated that mice can be reconstituted across both major and minor histocompatibility barriers with purified allogeneic hematopoietic stem cells (HSC). However, additional studies have demonstrated that non HSC cellular populations from whole bone marrow (WBM) have the potential to facilitate such engraftment. We have previously reported that WBM populations with facilitator potential are characterized by the surface marker profiles CD8+TCR- CD3- and CD8+TCR+. Current clinical protocols in bone marrow transplantation are attempting to decrease donor morbidity by utilizing methods of mobilizing pertinent cells into the peripheral blood for donor harvest. The studies described in this abstract were undertaken to evaluate the potential of CD8+ populations in mobilized peripheral blood for enhancing the engraftment of HSC across allogeneic barriers. When BALB/c mice are lethally irradiated and injected with 500 hematopoietic stem cells, 20% survive. Thirty BALB/c mice were irradiated with 900 rad TBI. Ten mice were given 500 purified HSC(c-Kit+Thy-1.1loLin-/loSca-1+) alone. Ten mice were given 500 HSC and 20,000 CD8+ cells. Six mice were given HSC and 25,000 CD8+TCR+ cells. Two mice were given HSC and 4000 CD8+TCR- cells. Two mice were given CD8+ cells alone. Survival at 17 days was significantly enhanced in mice receiving HSC and CD8+ TCR- cells (2/2), HSC and CD8+TCR+ cells (6/6), and HSC and CD8tot cells(10/10) when compared to mice that received HSC alone (2/10). The peripheral blood was evaluated in surviving mice at 6 weeks for evidence of donor-derived cells and lineage configuration. Surviving mice in groups that received CD8tot and CD8+TCR+ cells with HSC had significantly more donor derived cells (47% and 83% respectively) than control animals that received HSC alone(0.05%) . The enhancement of hematopoietic reconstitution extended to all hematopoietic lineages. These data demonstrate that facilitative potential is contained in the CD8+ population of mobilized peripheral blood as well as in WBM.