Patent Application: US-43159903-A

Abstract:
human pro - b cells deficient in pax5 expression and methods of producing them . human pax5 - deficient pro - b cells are useful for the therapy of disorders associated with a depletion of the lymphoid system , in particular for the treatment of immunodeficiencies like aids .

Description:
if not otherwise stated , the following materials and methods were used in the examples : pax5 +/− , pax5 f / f , rag2 −/− and creed - 30 mice were maintained on the c57bl / 6x129 / sv background and genotyped as described ( horcher et al ., 2001 ; urbanek et al ., 1994 ; schwenk et al ., 1998 ; shinkai et al ., 1992 ). b220 + pro - b cells were sorted from the bone marrow of pax5 +/− or pax5 f / f creed - 30 mice and cultured on y - irradiated st2 cells in il - 7 containing imdm medium as described ( nutt et al ., 1997 ). pax5 +/− pro - b cell clones were generated by sorting single cells from a cd19 − pax5 +/− pro - b cell pool with a facsvantage tso flow - cytometer ( becton - dickinson ). single - cell suspensions were stained and analysed on a facscalibur flow - cytometer ( becton - dickinson ) as described ( urbanek et al ., 1994 ). the following fluorescein isothiocyanate ( fitc )-, phycoerythrin ( pe )- or allophycocyanin ( apc )- labelled antibodies were purchased from pharmingen ( san diego , calif . ): anti - b220 ( ra3 - 6b2 ), anti - cd4 ( l3t4 ), anti - cd8 ( 53 - 6 . 7 ), anti - cd19 ( 1d3 ), anti - mac - i ( m1 / 70 ), anti - igm ( m41 . 42 ), anti - tcrβ ( h57 - 597 ) and anti - human cd2 ( rpa - 2 . 10 ) antibodies . the pe - anti - f4 / 80 ( c1 : a3 - 1 ) antibody were obtained from serotec ( oxford , england ). β - galactosidase activity was measured by loading the pro - b cells with the fluorogenic substrate 5 - chloromethylfluorescein di - β - d - galactopyranoside ( cmfdg , molecular probes ) as described ( nutt et al ., 1999c ). b220 + pro - b cells were sorted from the bone marrow of 4 - week - old pax5 f / f creed - 30 mice , expanded in vitro and then treated with 1 μm 4 - hydroxy - tamoxifen ( oht ; research biochemicals international , natick , mass .) for 3 days followed by subsequent culturing in the absence of oht . the cd19 − pax5 δ / δ pro - b cells were purified to homogeneity by eliminating the few non - deleting cd19 + pax5 f / f pro - b cells by facs sorting . the kinetic experiments were performed with freshly isolated pax5 f / f creed - 30 pro - b cells that were expanded ex vivo to 30 × 10 6 cells for only 9 days prior to oht treatment as described above . pro - b cells ( 6 . 5 × 10 6 ) were withdrawn at daily intervals after oht addition . dna isolated from 0 . 5 × 10 6 cells was analysed by pcr genotyping ( horcher et al ., 2001 ) to confirm deletion of pax5 exon 2 . total rna was prepared from 5 × 10 6 cells , using the trizol reagent ( gibco - brl , life technologies ). reverse transcription ( with random hexamer primers ) and semiquantitative pcr were performed as described ( horcher et al ., 2001 ; nutt et al ., 1999c ). gene seq id no : annealing ° c . cycles size ( bp ) igδ m 1 and 2 55 25 595 igμ m 3 and 4 55 25 513 j - chain 5 and 6 55 30 338 pax5 7 and 8 55 25 593 ( fl ) 427 ( δe2 ) hprt 9 and 10 55 25 313 ebf 11 and 12 55 30 481 m - csf - r 13 and 14 57 30 1200 cd19 15 and 16 59 25 847 mb - 1 17 and 18 59 30 820 b29 1 19 and 20 59 30 243 ciita 21 and 22 59 30 764 rest1 23 and 24 57 25 492 rest2 25 and 26 57 25 680 rest3 27 and 28 57 30 595 pax5 - lacz 29 and 30 55 25 611 the pcr products were separated on agarose gels and visualized by ethidium bromide staining . protein samples were prepared by directly dissolving and boiling 1 × 10 6 cells in 80 μl of 2 × sds sample buffer . total protein ( 10 μl ) was separated by 10 % sds - page and analyzed by western blotting with a rabbit polyclonal antibody directed against the pax5 paired domain ( adams et al ., 1992 ). pax5 δ / δ pro - b cells were cultured on st2 cells in the absence of il - 7 for ˜ 10 days followed by terminal differentiation in imdm medium containing m - csf ( 25 ng / ml ; r & amp ; d systems ). the phagocytic activity of differentiated cells was determined by incubation with fitc - labelled heat - inactivated e . coli ( molecular probes ) as described ( nutt et al ., 1999a ). rag2 −/− mice at 8 - 12 weeks of age were γ - irradiated with 550 rad and intravenously injected with 1 - 5 × 10 6 in vitro cultured pax5 δ / δ , pax5 f / f or pax5 +/− pro - b cells as described ( rolink et al ., 1999 ). prior to injection , the pax5 +/− pro - b cells were infected with the pmi retrovirus expressing a human cd2 protein lacking its intracellular domain ( deftos et al ., 1998 ). b - lineage commitment requires continuous expression of pax5 during early b cell development to address the question whether b - lineage commitment requires only the transient activation or continuous expression of pax5 during early b cell development , the inventors took advantage of the cre - loxp system for conditional inactivation of a floxed ( f ) pax5 f allele carrying loxp sequences on either side of the paired domain exon 2 ( horcher et al ., 2001 ). b cell development was normal in pax5 f / f mice , whereas pax5 δ / δ mice , generated by germline deletion ( δ ) of exon 2 ( horcher et al ., 2001 ), were phenotypically indistinguishable from pax5 −/− mice ( urbanek et al ., 1994 ). for conditional pax5 inactivation at the pro - b cell stage , pax5 f / f mice were crossed with the transgenic line creed - 30 , which expresses the cre - ebd [ g521r ] fusion protein under the control of the immunoglobulin eμ enhancer and sv40 promoter in the b - lymphoid lineage ( schwenk et al ., 1998 ). the cre - ebd [ g521r ] protein consists of the cre recombinase linked to a mutant human oestrogen receptor ligand - binding domain ( ebd ) that is insensitive to the natural ligand β - oestradiol , but is responsive to the synthetic antagonist 4 - hydroxy - tamoxifen ( oht ). this posttranslational induction system allows the specific induction of cre recombinase activity in b - lymphocytes by oht treatment ( schwenk et al ., 1998 ). therefore pro - b cells were sorted from the bone marrow of pax5 f / f creed - 30 mice and cultured these cells in vitro on stromal cells in the presence of il - 7 . flow cytometric analysis revealed that these pro - b cells express the b cell markers b220 and cd19 ( fig1 a , f / f ). as transcription of the target gene cd19 is strictly dependent on pax5 function ( nutt et al ., 1997 ; nutt et al ., 1998 ), these data demonstrate that the cd19 + pax5 f / f pro - b cells express normal levels of pax5 and have thus undergone commitment to the b - lymphoid lineage ( for further arguments , see below ). treatment of these pro - b cells with 1 μm oht resulted in the specific loss of cd19 expression , indicating that the creed - 30 transgene can be used for efficient inactivation of the floxed pax5 allele under the in vitro culture conditions used ( fig1 a , δ / δ ). it is important to note that the proliferation of the pro - b cells remained largely unaffected by the oht treatment ( data not shown ). hence , the oht - induced inactivation of pax5 did result neither in a significant cell loss nor in the outgrowth of a minor subpopulation of pax5 δ / δ pro - b cells . time course analyses revealed that pax5 f / f creed - 30 pro - b cells efficiently deleted the pax5 exon 2 within 1 day of oht treatment ( fig1 b ). within the same short period , the pro - b cells predominantly expressed the truncated pax5 transcript lacking exon 2 ( fig1 d ) in agreement with the fact that the pax5 mrna has a short half - life of ˜ 2 hours ( nutt et al ., 1999c ). in contrast , the more stable pax5 protein was reduced to a low level only 4 days after oht addition ( fig1 c ). next used rt - pcr analysis was used to investigate the gene expression changes caused by pax5 inactivation . one class consisted of the control genes , encoding the ubiquitous hprt mrna and the b - cell - specific ebf , b29 ( igβ ) and μ m transcripts , which were neither differentially expressed in wild - type and pax5 −/− pro - b cells nor affected by conditional pax5 loss ( fig1 d ). in contrast , the expression of target genes , which are normally activated by pax5 in pro - b cells , started to decline at day 4 , concomitant with the loss of pax5 protein ( fig1 d ). these target genes code for the cell surface protein cd19 and igα ( mb - 1 ) ( nutt et al ., 1998 ), the transcription factor ciita ( horcher et al ., 2001 ) and the alternatively spliced δ m transcript of the immunoglobulin igh gene ( horcher et al ., 2001 ). conversely , the genes that are normally silenced by pax5 at the onset of b cell development were derepressed upon pax5 inactivation ( fig1 d ). this class of genes codes for the secreted j - chain ( rinkenberger et al ., 1996 ), the m - csf receptor ( nutt et al ., 1999a ) and transcripts of unknown function . hence , the expression pattern of pax5 f / f pro - b cells appears to be indistinguishable from that of pax5 +/+ pro - b cells , but can be converted by pax5 inactivation into that of pax5 −/− pro - b cells . it was therefore concluded that the pax5 - dependent gene expression program is reversible in pro - b cells undergoing pax5 inactivation . pax5 - deficient pro - b cells adopt again the broad developmental potential of early hematopoietic progenitor cells as the pax5 δ / δ pro - b cells were directly derived from committed pax5 f / f pro - b cells , it was next investigated whether the pax4 δ / δ pro - b cells remained committed to the b cell pathway or adopted again the broad developmental potential of early hematopoietic progenitor cells . the potential of the pax5 δ / δ pro - b cells to differentiate into macrophages was assessed by culturing them in the absence of il - 7 on stromal st2 cells , which abundantly produce the myeloid cytokine m - csf ( nutt et al ., 1999a ). pax5 δ / δ cells , that were differentiated under these conditions for up to 3 weeks , down - regulated the b cell surface protein b220 , expressed the macrophage markers mac - 1 and f4 / 80 ( fig2 a ) and displayed the characteristic morphology of enlarged vacuolar macrophages ( fig2 b ). these differentiated cells furthermore phagocytosed fluorescently labeled e . coli ( fig2 c ), indicating that the pax5 δ / δ pro - b cells can differentiate into mature macrophages like the pax5 −/− pro - b cells ( nutt et al ., 1999a ). pax5 deficient pro - b cells have the potential to develop into t - lymphocytes the potential of pax5 δ / δ pro - b cells to develop into t - lymphocytes was analyzed by intravenous injection into sublethally irradiated rag2 −/− mice , which have an early block in t and b cell development due to the absence of v ( d ) j recombination ( shinkai et al ., 1992 ). three weeks after cell transfer , the cellularity of the thymus was considerably increased due to the presence of cd4 + cd8 + ( double - positive ) as well as cd4 + cd8 − and cd8 + cd4 − ( single - positive ) thymocytes ( fig3 a ). these thymocyte subpopulations were derived from the injected pax5 δ / δ pro - b cells , as they expressed the t cell receptor ( tcr ) β similar to wild - type thymocytes ( fig3 a ). hence , the pax5 δ / δ pro - b cells could fully reconstitute t cell development , but failed to generate igm + b cells in the spleen of rag2 −/− mice ( fig3 b ) in analogy to pax5 −/− pro - b cells ( rolink et al ., 1999 ). the opposite situation was observed for the pax5 f / f pro - b cells , which failed to give rise to tcrβ + thymocytes , but developed into igm + b cells in the spleen of injected rag2 −/− mice ( fig3 c ). hence , the pax5 f / f pro - b cells are fully committed to the b cell pathway . it was therefore concluded that the inactivation of pax5 alone is sufficient to revert the narrow b - lymphoid potential of pax5 f / f pro - b cells to the broad multilineage potential characteristic of early hematopoietic progenitor cells . decommitment of pax5 +/− pro - b cells upon switching of expression from the wild - type to the targeted pax5 allele the heterozygous pax5 +/− mouse provided a second opportunity to investigate the reversibility of pax5 - dependent b - lineage commitment , as the pro - b cells of this mouse preferentially express only one of the two pax5 alleles ( nutt et al ., 1999c ). interestingly , heterozygous cd19 + pro - b cells are able to switch expression from the wild - type pax5 allele to the targeted pax5 lacz (−) allele during in vitro culture , which may reflect a proliferative advantage of the pax5 null phenotype for the in vitro propagation of pro - b cells ( nutt et al ., 1999b ; nutt et al ., 1999c ). this allele switching results in the loss of expression of the pax5 target gene cd19 and simultaneous activation of the lacz gene of the targeted pax5 locus ( nutt et al ., 1999b ; nutt et al ., 1999c ) ( fig4 a ). therefore , cd19 − β - gal + pro - b cell clones were derived from pax5 +/− bone marrow ( fig4 a ) and demonstrated by rt - pcr analysis that these pro - b cells switched off the expression of activated pax5 target genes and derepressed genes normally suppressed by pax5 ( fig4 b ; data not shown ). hence , the cd19 − β - gal + pro - b cells apparently reversed their gene expression program to that of homozygous pax5 −/− cells , although , as genotypically heterozygous cells , they still have the ability to reactivate the wild - type pax5 allele . these cd19 − pax5 +/− pro - b cells were also decommitted , as they differentiated , upon il - 7 withdrawal into functional mac - 1 + f4 / 80 + macrophages with high phagocytic activity , similar to pax5 −/− cells ( fig4 c , d ; data not shown ). to study the t - lymphoid potential , the cd19 − pax5 +/− pro - b cells were first infected with a human ( h ) cd2 - expressing retrovirus prior to injection into sublethally irradiated rag2 −/− mice . three weeks after cell transfer , the thymus contained cd4 + cd8 + , cd4 + cd8 − and cd8 + cd4 − t cells of donor origin ( hcd2 + ), indicating that the cd19 − pax5 +/− pro - b cells are also able to reconstitute t cell development ( fig4 e ). thymic reconstitution was still observed 11 weeks after cell transfer due to long - term engraftment of the cd19 − b220 + pax5 +/− pro - b cells in the bone marrow , from where these progenitors continuously seed the thymus ( fig4 f , data not shown ). in addition to these cd19 − b 220 + pro - b cells , the bone marrow contained cd19 + b220 + b - lymphocytes of donor origin ( hcd2 + ), which have re - entered the b cell pathway due to activation of the wild - type pax5 allele ( fig4 f ). finally , the bone marrow cells also contained a third hcd2 + cd19 − b220 − cell population ( fig4 f ), which primarily consisted of mature cd4 + and cd8 + t - lymphocytes ( data not shown ). in summary , heterozygous pax5 +/− pro - b cells did not only regain the broad developmental potential of early hematopoietic progenitors upon switching to the mutant pax5 allele , but at the same time maintained their competence to undergo b cell development by reactivating the wild - type pax5 allele in vivo . low - density mononuclear cells are prepared from human umbilical cord blood and enriched for cd34 + progenitor cells by macs sorting with cd34 immunomagnetic beads ( miltenyi , auburn , calif .). the bead - sorted cd34 + cells ( 40 - 80 % pure ) are then stained with cychrome - labeled anti - cd34 , fluorescein isothiocyanate - coupled anti - cd10 and phycoerythrin - labeled anti - cd19 antibodies , and the cd34 + cd19 − cd10 − cells are sorted using a facsvantage tso flow - cytometer ( becton - dickinson ). the purified cd34 + cd19 − cd10 − cells are cultured in a pro - b cell medium on petri dishes that are precoated with a confluent γ - irradiated monolayer of murine ms - 5 stromal cells ( berardi et al , 1997 ). the pro - b cell medium consists of iscove &# 39 ; s dulbecco modified medium ( imdm ) containing 10 % human ab serum ( institut jacques boy , reims , france ), 5 % pre - tested fetal calf serum ( hcc - 6400 ; stem cell technologies inc ., vancouver , canada ) and the following recombinant ( r ) human ( hu ) cytokines : rhu - 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