cd8 and cd4   t cell subsets were isolated from heparinized umbilical cord   blood as described ( 10 ) . in brief ,
mononuclear cells obtained by   centrifugation on ficoll - metrizoate gradients were treated with   l - leucine methyl ester to remove monocytes and nk cells ; enriched t cells were isolated by treating cells forming rosette with   srbcs by means of lympho - kwik t ( one lambda , canogalark , ca ) . cd8 t cells were positively selected with dynabeads m-450 cd8 ( dynal , great neck , ny ) and further depleted of cd4 cells with dynabeads m-450 cd4 ; cd4 t cells   were obtained by treating enriched t cells with lympho - kwik t   helper ( one lambda ) .
neonatal t cell subsets were > 98% cd3   and < 1% cd14 , cd20 , cd56 , or cd16 positive , respectively ;   the cd8 subset was > 95% tcr-/ cd8-/ , > 99%   cd45ra / ro , and contained no detectable cd4 or cd29   positive cells .
t cells were submitted to one or two cycles of activation and il-2 expansion as described ( 11 ) . at each cycle , t   cells were activated with anti - cd3 mab ( ucht-1 , 200 ng / ml ;   a gift from p. beverley , university college and middlesex school   of medicine , london , uk ) and irradiated cd32 , b7.1 transfected l cells for 3 d , and cells were then washed twice and expanded in fresh medium supplemented with 50 iu / ml of il-2 for   4 d. t cells were then washed and either stored in liquid nitrogen , until use for hiv infection , or subjected to another cycle of   activation / expansion . in some experiments , cd8 t cells ( 5  10 cells / ml ) were   stimulated with irradiated allogeneic dendritic cells ( 5  10   cells / ml ) in the presence of il-2 ( 50 iu / ml ) .
dendritic cells were   derived from adult blood monocytes cultured for 9 d with il-4 ,   gm - csf , and tnf- exactly as described ( 12 ) .
one- or two - color flow cytometric analysis   was performed on a facsort ( becton dickinson , montreal ,   canada ) according to standard procedures .
the following fitc-   or pe - labeled mabs or isotype - matched mouse ig were purchased from becton dickinson : anti  cd8- ( leu 2a ) , anti - cd4   ( leu 3a ) , anti - cd3 , anti - cd20 , anti - cd14 , anti - cd56 , and anti - cd16 .
cd4 was also stained with okt4 ( american type culture   collection , rockville , md ) ; mab to cd8-/ heterodimer   ( 2st8 ) was given by e. reinherz ( harvard medical school , boston , ma ) ; the anti - cxcr4/fusin mab ( 12g5 ) has been described ( 13 ) .
total cellular rna was extracted   from resting or activated cord blood cd4 or cd8 t cells using rna ease total rna kit ( qiagen , chasworth , ca ) and pretreated with 10 u / ml rnase - free dnase ( gibco brl , gaithersburg , md ) before amplification .
synthesis of cdna and ensuing pcr amplification were performed using titan reverse   transcriptase ( rt)-pcr kit ( boehringer mannheim , indianapolis ,   in ) according to the supplier 's instruction manual .
pcr products   were separated on a 1% agarose gel and stained with ethidium   bromide .
chinese hamster ovary cell rna served as negative control for cd4 , cxcr4 , and ccr5 genes ; glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) was amplified to confirm equal   efficiency of amplification of each rna .
no pcr product was   detected in control amplification containing no added rna , taq ,   or rt dna polymerase .
primers for amplification were as follows :   cxcr4 : 5-agaattcaaccagcggttaccatggaggggatc , 5-agaattcgtcttttacatctgtgttagctggag ; ccr5 : 5-tcgatccggtggaacaagatggattatcaag , 5-tcggatccaagccatgtgcacaactctgactg ; and cd4 : 5-gcagtggcgagctgtggt , 5-gggtccccacacctcacagg .
either freshly activated or cryopreserved neonatal cd4 and cd8 cells were infected with hiv-1us-1 or hiv-1nl4 - 3 as previously described ( 14 ,   15 ) . in brief , for each infection 5  10 cells were centrifuged ,   washed , and resuspended in 400 l of media containing 13 x 10   tissue culture and infectious dose ( tcid)50 of hiv-1 .
the cells   were incubated at 37c for 2 h , washed three times to remove   excess virus , and resuspended in 50% conditioned media at 10/ml .   at t = 0 , 2 , 72 , and 144 h after infection
the cell pellets were lysed , amplified by pcr using hiv gag - specific primers , and the amplified   sequences were detected by hybridization to a radiolabeled oligonucleotide specific for internal gag sequences .
the hybridized   products were separated by gel electrophoresis and exposed to a   phosphorimager screen for 1 h. to ensure that the reactions were   performed within the linear range of the assay , log increments   hiv gag plasmid standards were amplified at the same time . to   show that equivalent levels of input dna were present in each   pcr reaction ,
human -globulin sequences were pcr amplified   as described ( 15 ) . after washes 12  10   cells were stained with optimized concentrations of cell surface   marker antibodies for 1 h at 4c .
the cells were washed twice   with sterile pbs , ph 7.4 , and fixed in 100 l of permeafix   ( ortho diagnostics , raritan , nj ) for at least 1 h at room temperature .
cells were washed in sterile pbs , ph 7.4 , pelleted by centrifugation , and then washed again in 2 standard saline citrate   ( ssc ) .
after centrifugation , the cell pellet was resuspended in hybridization solution ( 2 ssc , 30% formamide , sonicated salmon   sperm , yeast transfer rna ) containing 500 ng of 5-carboxy - fluorescein double - end  labeled , gag - pol 
specific oligonucleotide probes   or gag - pol sense oligonucleotides as a negative control probe   cocktail ( 16 ) .
the intracellular hybridization was performed at   42c for 1 h followed by successive washes in 2 ssc , 0.5% triton x-100 , and 1 ssc ,
the cells   were resuspended for analysis in pbs , ph 8.3 , and analyzed on a   flow cytometer ( epic xl ; coulter , miami , fl ) .
cd8 and cd4   t cell subsets were isolated from heparinized umbilical cord   blood as described ( 10 ) . in brief ,
mononuclear cells obtained by   centrifugation on ficoll - metrizoate gradients were treated with   l - leucine methyl ester to remove monocytes and nk cells ; enriched t cells were isolated by treating cells forming rosette with   srbcs by means of lympho - kwik t ( one lambda , canogalark , ca ) . cd8 t cells were positively selected with dynabeads m-450 cd8 ( dynal , great neck , ny ) and further depleted of cd4 cells with dynabeads m-450 cd4 ; cd4 t cells   were obtained by treating enriched t cells with lympho - kwik t   helper ( one lambda ) .
neonatal t cell subsets were > 98% cd3   and < 1% cd14 , cd20 , cd56 , or cd16 positive , respectively ;   the cd8 subset was > 95% tcr-/ cd8-/ , > 99%   cd45ra / ro , and contained no detectable cd4 or cd29   positive cells .
t cells were submitted to one or two cycles of activation and il-2 expansion as described ( 11 ) . at each cycle , t   cells were activated with anti - cd3 mab ( ucht-1 , 200 ng / ml ;   a gift from p. beverley , university college and middlesex school   of medicine , london , uk ) and irradiated cd32 , b7.1 transfected l cells for 3 d , and cells were then washed twice and expanded in fresh medium supplemented with 50 iu / ml of il-2 for   4 d. t cells were then washed and either stored in liquid nitrogen , until use for hiv infection , or subjected to another cycle of   activation / expansion . in some experiments , cd8 t cells ( 5  10 cells / ml ) were   stimulated with irradiated allogeneic dendritic cells ( 5  10   cells / ml ) in the presence of il-2 ( 50 iu / ml ) .
dendritic cells were   derived from adult blood monocytes cultured for 9 d with il-4 ,   gm - csf , and tnf- exactly as described ( 12 ) .
one- or two - color flow cytometric analysis   was performed on a facsort ( becton dickinson , montreal ,   canada ) according to standard procedures .
the following fitc-   or pe - labeled mabs or isotype - matched mouse ig were purchased from becton dickinson : anti 
cd8- ( leu 2a ) , anti - cd4   ( leu 3a ) , anti - cd3 , anti - cd20 , anti - cd14 , anti - cd56 , and anti - cd16 .
cd4 was also stained with okt4 ( american type culture   collection , rockville , md ) ; mab to cd8-/ heterodimer   ( 2st8 ) was given by e. reinherz ( harvard medical school , boston , ma ) ; the anti - cxcr4/fusin mab ( 12g5 ) has been described ( 13 ) .
total cellular rna was extracted   from resting or activated cord blood cd4 or cd8 t cells using rna ease total rna kit ( qiagen , chasworth , ca ) and pretreated with 10 u / ml rnase - free dnase ( gibco brl , gaithersburg , md ) before amplification .
synthesis of cdna and ensuing pcr amplification were performed using titan reverse   transcriptase ( rt)-pcr kit ( boehringer mannheim , indianapolis ,   in ) according to the supplier 's instruction manual .
pcr products   were separated on a 1% agarose gel and stained with ethidium   bromide .
chinese hamster ovary cell rna served as negative control for cd4 , cxcr4 , and ccr5 genes ; glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) was amplified to confirm equal   efficiency of amplification of each rna .
no pcr product was   detected in control amplification containing no added rna , taq ,   or rt dna polymerase .
primers for amplification were as follows :   cxcr4 : 5-agaattcaaccagcggttaccatggaggggatc , 5-agaattcgtcttttacatctgtgttagctggag ; ccr5 : 5-tcgatccggtggaacaagatggattatcaag , 5-tcggatccaagccatgtgcacaactctgactg ; and cd4 : 5-gcagtggcgagctgtggt , 5-gggtccccacacctcacagg .
either freshly activated or cryopreserved neonatal cd4 and cd8 cells were infected with hiv-1us-1 or hiv-1nl4 - 3 as previously described ( 14 ,   15 ) . in brief , for each infection 5  10 cells were centrifuged ,   washed , and resuspended in 400 l of media containing 13 x 10   tissue culture and infectious dose ( tcid)50 of hiv-1 .
the cells   were incubated at 37c for 2 h , washed three times to remove   excess virus , and resuspended in 50% conditioned media at 10/ml .   at t = 0 , 2 , 72 , and 144 h after infection
the cell pellets were lysed , amplified by pcr using hiv gag - specific primers , and the amplified   sequences were detected by hybridization to a radiolabeled oligonucleotide specific for internal gag sequences .
the hybridized   products were separated by gel electrophoresis and exposed to a   phosphorimager screen for 1 h. to ensure that the reactions were   performed within the linear range of the assay , log increments   hiv gag plasmid standards were amplified at the same time . to   show that equivalent levels of input dna were present in each   pcr reaction , human -globulin sequences were pcr amplified   as described ( 15 ) .
after washes 12  10   cells were stained with optimized concentrations of cell surface   marker antibodies for 1 h at 4c .
the cells were washed twice   with sterile pbs , ph 7.4 , and fixed in 100 l of permeafix   ( ortho diagnostics , raritan , nj ) for at least 1 h at room temperature .
cells were washed in sterile pbs , ph 7.4 , pelleted by centrifugation , and then washed again in 2 standard saline citrate   ( ssc ) .
after centrifugation , the cell pellet was resuspended in hybridization solution ( 2 ssc , 30% formamide , sonicated salmon   sperm , yeast transfer rna ) containing 500 ng of 5-carboxy - fluorescein double - end  labeled , gag - pol  specific oligonucleotide probes   or gag - pol sense oligonucleotides as a negative control probe   cocktail ( 16 ) .
the intracellular hybridization was performed at   42c for 1 h followed by successive washes in 2 ssc , 0.5% triton x-100 , and 1 ssc ,
the cells   were resuspended for analysis in pbs , ph 8.3 , and analyzed on a   flow cytometer ( epic xl ; coulter , miami , fl ) .
highly purified umbilical cord blood cd8 t cells   ( > 98% cd3 , > 95% tcr-/ , cd8-/ , no detectable cd4 cells ) and cd4 t cells ( > 98% cd3   cd4 , no detectable cd8 cells ) were activated with   anti - cd3 mab cross - linked on cd32 and b7.1 transfected   l cells , expanded in il-2supplemented medium , and then   infected with the macrophage - tropic hivus-1 or with the   t cell  tropic ( t - tropic ) hivnl4 - 3 virus ; hiv replication   was evaluated at days 3 and 6 after infection by quantitative   pcr analysis of gag dna accumulation .
both cd8 and   cd4 t cell subsets supported significant replication of the   macrophage tropic ( m - tropic ) , but not the t - tropic , strain   of hiv ( fig .
1 ) , although the initial infection was slower in   cd8 than in cd4 t cells , as judged from the day 3 values . that hiv infected cd8 t cells , and not contaminating cd4 cells was supported by 3 lines of evidence . first
after infection with hivus-1 for 6 d ,   cd8 t cells were surface stained for cd8 expression and   stained intracellularly for hiv gag and pol rna sequences .  
in addition , to confirm that a spreading infection   was occurring in the neonatal cd8 t cells , samples were   collected on both days 3 and 6 after infection and analyzed .   at day 3 ,
3.4% of the cells were double positive for cd8   and hiv rna , and by day 6 the number grew to 12.3% ,   which is consistent with a productive infection ( data not   shown ) .
second , cd8 t cell cultures did not contain detectable cd4 single positive cells at day 6 after infection   ( data not shown ) .
third , cd4 ag and cd4 messenger   rna ( mrna ) were undetectable in freshly prepared   cd8 t cells , as assessed by flow cytometry and rt - pcr   analysis respectively ( fig .
3 , a and b ) . thus , activated neonatal cd8 t cells can be productively infected by primary   m - tropic hiv isolates , responsible for disease transmission ,   but not by t - tropic hiv .
the apparent resistance of neonatal cd4 and cd8 t cell subsets to t - tropic hiv   could be related to the recent finding that anti - cd3/b7 activated naive cd4 t cells isolated from adult blood are   also resistant to t - tropic hiv , as a result of postentry block   in viral replication ( 1720 ) .     to be susceptible to hiv infection
, a cell must express   cd4 , the primary hiv receptor , together with either   ccr5 ( the coreceptor for m - tropic strains ) , or cxcr-4/ fusin ( the coreceptor for t - tropic strains ) ( for review see   reference 21 ) . according to this paradigm ,
indeed , we found that neonatal single positive   cd8 t cells started to express low to moderate levels of   cd4 within 24 h after anti - cd3/b7.1 activation ( fig .
3 a ) ; cd4 expression reached a plateau at 72 h ( 60 to > 90%   double positive cells , n = 24 ) and remained stable for at   least 2 wk ( the duration of the present experiments ) .
flow   cytometric analysis of anti - cd3/b7.1stimulated cd8 t   cells stained with anti - cd4 revealed a unimodal histogram ,   suggesting that the ability to coexpress cd4 is a common   property of most , if not the entire , neonatal cd8 t cell   population ( fig .
quantitatively , cd4 was expressed   at significantly lower levels on activated cd8 t cells than   on cd4 single positive t cells ( fig .
rt - pcr analysis revealed that cd4 mrna was undetectable in freshly   isolated cd8 t cells , but clearly induced after anti - cd3/ b7.1 stimulation , suggesting transcriptional activation of   the cd4 gene ( fig .
3 b ) . in experiments designed to determine the minimal requirements for cd4 expression on   neonatal cd8 t cells
, we found that : ( a ) tcr / cd3-mediated signals were sufficient to induce low levels of cd4 ,   but that cd4 induction was markedly enhanced by cd28   costimulation ( fig .
3 d ) , and ( b ) cellular proliferation was   neither sufficient or necessary .
indeed , cd4 was not expressed on proliferating cd8 t cells stimulated with anti - cd2 together with anti - cd28 mabs and il-2 , whereas it   was induced on irradiated ( nonproliferating ) anti - cd3/ b7.1stimulated cells ( data not shown ) .
finally and importantly , cd4 was coexpressed on neonatal t cells activated   under more physiological conditions , i.e. , by antigenic   stimulation with allogeneic dendritic cells ( fig .
thus ,   physiological activation of umbilical cord blood cd8 t   cells is associated with the coexpression of low to moderate   levels of cd4 , most likely as a result of cd4 gene transcriptional activation . moreover ,
effective costimulation is   required for optimal induction of cd4 coexpression , presumably explaining why this phenomenon went unnoticed   in earlier studies .
consistent with their susceptibility to infection with m - tropic hiv , activated neonatal t cells expressed ccr5 mrna ( fig .
as   already mentioned , it is suggested that their resistance to   t - tropic hiv infection results from a postentry block in   viral replication ( 1720 ) .
the ability to coexpress cd4 is   not a unique feature of neonatal cd8 t cells , and indeed   a variable proportion ( 1040% , n = 5 ) of cd8 t cells isolated from adult blood expressed low levels of cd4 after   anti - cd3/b7.1 activation ( data not shown ) .
since neonatal   cd8 t cells are immunologically naive whereas their   adult counterparts contain both naive and memory cells , it   will be of interest to examine cd4 expression and susceptibility to hiv infection in naive and memory cd8 t cell   subsets .
preliminary results suggest that indeed , naive but   not memory adult cd8 t cells may be induced to coexpress cd4 and become infectable by hiv .
they challenge the paradigm that   the cd4 gene is irreversibly silenced in extrathymic mature   single positive cd8-/ tcr-/ t lymphocytes and   raise the question of the role of cd4 in the biology of   cd8 t cells . a small fraction ( 23% ) of peripheral tcr-/ t cells
are cd8 cd4 double positive ( 22 ) . in the   large majority of the cases , these cells are cd4 cd8   and express cd8 as a cd8-/ homodimer ( 23 ) . in three   cases
however , the double positive t cells were shown to   display the same phenotype as activated neonatal cd8 t   cells , i.e. , cd8-/ cd4 , indicating that cells expressing this phenotype exist in vivo ( 24 ) .
finally , infection   of cd8 t cells by human herpesvirus 6 was previously   shown to induce cd4 expression and confer susceptibility   to hiv infection ( 25 ) . from a clinical point of view
, our   finding may explain the recent reports that cd8 t cells of   infected patients can harbor hiv-1 ( 26 , 27 ) .
it is tempting   to relate the hiv susceptibility of neonatal cd8 t cells to   the rapid progression of the disease in a large proportion of   infected neonates . since viral replication
is mainly controlled by cd8 t cells , it seems reasonable to assume that   the infection of these cells may compromise the immune   response to hiv .
productive infection of primary hiv - specific cd8 t cells may lead to an early disruption of the   ag repertoire of hiv - specific cd8 t cells .
infected   cd8 t cells may be deleted directly , after supporting   high rates of viral replication , or indirectly by cytotoxic   hiv - specific cd8 t cells .
early restriction of the antigen   repertoire would facilitate the emergence of hiv variants   expressing functionally important ags that were recognized   by the deleted t cells ( 2830 ) .
future studies aiming to   verify in vivo some of these hypotheses and to confirm the   differential susceptibility of naive versus memory cd8 t   cells to hiv infection should further our understanding of   the disease and have important implications for the development of hiv vaccines .
infection of neonatal cd4 and cd8 cells . umbilical cord   blood cd4 and cd8 t cells were activated with anti - cd3 and b7 - 1transfected cd32 l cells for 3 d , washed , expanded in il-2 , and infected with either a ccr5-dependent virus , hivus1 , or a cxcr4-dependent virus , hiv-1nl4 - 3 .
the phosphorimage shows the accumulation of   gag dna in cd4 and cd8 cultures at 0 , 2 , 72 , and 144 h after infection .   to ensure equivalent amounts of dna
neonatal cd8 t cells were harvested 6 d after infection and stained with both   anti - cd8 and labeled hiv dna probes .
left , facs analysis of cells infected with hivus1 ; right , a mock - infected control .
umbilical cord blood cd8 t cells were activated with anti - cd3   mab immobilized on b7.1 and cd32transfected l cells and expanded in il-2supplemented medium .
( a ) two - dimensional contour plot showing expression of cd4 and cd8 before t cell activation , after 24 and 72 h of primary activation , and at the end of a second cycle of activation / il-2 expansion   ( day 14 ) .
similar results were obtained by staining with okt4 mab , recognizing another cd4 epitope than leu3a mab .
more than 95% of resting or   activated cd8 t cells were stained brightly with 2st8 mab ( received from e. reinherz , harvard medical school , boston , ma ) specific for the cd8-/ heterodimer .
( b ) expression of cd4 , cxcr4 , and ccr5 mrna in resting and activated ( day 7 ) neonatal cd4 and cd8 t cells .
cells were stained with anti - cd4 mab at the end of the first cycle of activation / il-2   expansion ( day 7 ) .
( d ) cd4 expression on cd8 t cells activated with either plate - coated anti - cd3 ( 10 g / ml ) , immobilized anti - cd3 together with   soluble anti - cd28 ( clone 9.3 , 500 ng / ml ) , anti - cd3 ( 200 ng / ml ) immobilized on b7.1 cd32 l cells , or allogeneic dendritic cells together with il-2 ( 50   iu / ml ) .
cells were stained at day 7 with anti - cd4 and anti - cd8 mabs .
anti - cd3/b7.1activated   cd8 t cells were stained with 12g5 mab at day 7 .
 cd8 + t lymphocytes confer significant but ultimately insufficient protection against hiv infection . here 
 we report that activated neonatal cd8 + t cells can be productively infected in   vitro by macrophage - tropic ( m - tropic ) hiv-1 isolates , which are responsible for disease   transmission , whereas they are resistant to t cell  tropic ( t - tropic ) hiv strains . 
 physiological   activation of cd8-/+ cd4 t cell receptor/+ neonatal t cells , including activation by   allogeneic dendritic cells , induces the accumulation of cd4 messenger rna and the expression of cd4 ag on the cell surface . the large majority of anti - cd3/b7.1activated cord blood   cd8 + t cells coexpress cd4 , the primary hiv receptor , as well as ccr5 and cxcr4 , the   coreceptors used by m- and t - tropic hiv-1 strains , respectively , to enter target cells . 
 these   findings are relevant to the rapid progression of neonatal hiv infection . 
 infection of primary   hiv - specific cd8 + t cells may compromise their survival and thus significantly contribute to   the failure of the immune system to control the infection . 
 furthermore , these results indicate a   previously unsuspected level of plasticity in the neonatal immune system in the regulation of   cd4 expression by costimulation . 