pemphigus vulgaris ( pv ) is a life - threatening autoimmune blistering disease mediated by autoantibodies ( autoabs ) directed against desmogleins ( dsg ) located on the surface of keratinocyte cells ( kc ) . this leads to an intraepithelial loss of adhesion called acantholysis , and clinically it presents with vescicles and blisters . autoabs in pv are directed mainly against desmoglein 3 ( dsg 3 ) , a desmosomal glycoprotein situated in the skin predominantly in the suprabasilar epidermal layer , and less frequently against desmoglein 1 . though the pathogenetic role of antidesmoglein autoabs is certain , the exact mechanism through which they lead to acantholysis is still incompletely understood . complement , plasminogen - plasmin , cytokines , cell - mediated immunity , and other autoantibodies such as anticholinergic receptor antibodies have been suggested in determining acantholysis in pv . studies conducted so far regarding the role of t cells involved mainly cd4 + lymphocytes for their cooperation with b cells and subsequently for the induction and regulation of autoabs production . the function of cd8 + t cells has not been explored yet , but some authors hypothesize their role in cell - mediated pathogenesis of pv . other studies suggested a possible role of natural killer ( nk ) cells   as well as fas and caspase 8 in pv . fas is a member of the tumor necrosis factor ( tnf ) receptor family that is bound by fas ligand , expressed on t cd8 + cells . in this study we sought to evaluate the role of cd8 + cells performing a passive transfer of pv autoabs using cd8 deficient mice ( cd8/ ) . the results of these studies suggest a role for cd8 in the pathogenesis of pv . immunohistochemical staining was performed using the alkaline phosphatase - antialkaline phosphatase ( apaap ) method on 7  m skin sections of 7 pv patients . monoclonal antibodies against cd3 ( 1  :  20 ; dako , glostrup , denmark ) , cd4 ( 1  :  20 ; dako ) , and cd8 ( 1  :  20 ; dako ) were used . for the quantitative study , stained cells were first counted in three consecutive microscopic fields ( 250x ) , both in the dermis and in the epidermis , and then summed ; the average value was then calculated . plasma was obtained from the plasmapheresis of one patient with clinical , histologic , and immunologic features consisting of the diagnosis of pv , during the acute phase of the disease . total igg concentration was measured by nephelometry using monospecific goat anti - human igg ( beckman instruments , missasauga , on , canada ) . pemphigus ab titers were measured by indirect immunofluorescence ( iif ) using monkey esophagus epithelium as the tissue substrate . as a negative control , igg fractions were isolated and removed from pv plasma using protein a ( pa ) . isolation of igg fractions from pv plasma was achieved by standardized technique using staphylococcal protein a coupled to sepharose 4b   ( pharmacia biotech , uppsala , sweden ) . pa was washed four times in cold pbs and finally incubated with pv plasma overnight at 4c . absence of igg fractions in the control plasma was assessed by iif staining on a monkey esophagus epithelium substrate and confirmed by nephelometry . pv plasma and control plasma were filter sterilized with millex ( pore size 0.22  mm ; millipore , bedford , ma ) and stored at 20c . all mice were housed and bred under specific pathogen - free conditions in the animal facility of the sunnybrook health science centre . an average of 15 mice within each experimental group was used , and each experiment was repeated at least three times . all animal procedures were approved by the sunnybrook health science centre animal care committee . 
 cd8 
 / 
  mice . the generation of mice homozygous for cd8 gene mutations ( cd8/ ) was obtained by disruption of the lyt-2 gene through homologous recombination , and the mutation was interbred into the c57bl/6 background before generating cd8-deficient ( cd8/ ) mice . mice homozygous for the defect were used as the knockout ( ko ) mice , with the wild - type ( wt ) animals serving as the nondeficient controls . to induce pv in mice briefly , plasma was injected intradermally , in the dorsal area , into neonatal mice through a 30-gauge needle . the total dose administered ranged from 30 to 50  l / g of body weight in a single administration . we chose a dose of 30  l / g because this was the minimum dose inducing the disease in wt mice . cd8/ mice and c57bl/6 mice were injected with the same dose of pv plasma . as a negative control , gene targeted mutant mice and wt mice were injected with plasma depleted of igg by treatment with protein a. mice were examined 24  h after the injections . lesional and perilesional skin were obtained for light microscopy and direct immunofluorescence ( dif ) 24  h after injection with pv igg . at the time of biopsies , serum was also obtained and assayed for iif on a monkey esophagus epithelium to detect the igg titer . cryostat sections ( 5  m ) were used , and dif studies were performed according to standard techniques . briefly , specimens were washed in pbs for 10 minutes , incubated for 30 minutes with fitc - conjugated f(ab)2 fragment of rabbit anti - human igg , specific for -chains ( 1  :  25 ; dako , glostrup , denmark ) , and washed in pbs for 15 minutes . slides were covered with buffered glycerol , and results were read in a nikon optiphot immunofluorescence microscope ( nikon , melville , ny ) . sera were collected 24  h after pv igg treatment or pa treatment ( igg depleted ) , and iif studies were performed according to standard techniques . cryostat sections ( 5  m ) of monkey esophagus were used as substrate , washed for 10  min in pbs , incubated for 30  min with different concentrations of sera ( 1  :  1 - 1  :  600 ) , washed in pbs for 15  min , labeled with fitc - conjugated f(ab)2 fragment of rabbit anti - human igg ( dako , glostrup , denmark ) for 30  min , and then washed again in pbs for 10  min . slides were covered with buffered glycerol , and results were examined using a nikon optiphot immunofluorescence microscope ( nikon , melville , ny ) . skin biopsies from mice were fixed in 10% formalin and stained with hematoxylin and eosin . data regarding the incidence of the disease in ko and control mice were analyzed using the  test ; a p value < 0.05 was considered to be significant . cd3 + t cells were detected both in the dermis ( 32.8  1.6 cells counted as mentioned in section 2 ) , with a perivascular distribution , and in the epidermis ( 7.9  2.8 ) of all patients . cd4 + t cells were found in the superficial and papillary dermis ( 33.6  4.8 ) with scattered and perivascular distribution , and a fewer number of them were detected in the basal and suprabasal layers near the dermal - epidermal junction ( 4.2  1.2 ) . a number of cd8 + t cells ( 14.2  1.6 ) were observed in perivascular areas of dermal lesional skin ( cd4/cd8 = 2.7 ) ( table 1 ) . iif for pv igg using monkey esophagus as substrate demonstrated a titer of 1  :  2460 , and an igg concentration of 5.9  mg / ml was measured by nephelometry . pa - treated plasma showed absence of intercellular staining on monkey esophagus , and igg levels were below the level of detection using the nephelometric analysis . the passive transfer of pv igg demonstrated a direct correlation between the amount of pv igg injected and the incidence of the disease . acantholysis and inflammatory infiltrate were evident in mice given plasma containing pv igg and absent in mice injected with igg - depleted plasma . the epidermis of mice injected with pv igg who developed pv lesions showed human igg bound to the intercellular cell surface by dif . no difference was observed in the intensity of fluorescence in different strains of mice treated with an equal dose of pv igg . the igg titer in all pv plasma injected mice detected by iif ranged between 1  :  100 in mice treated with 30  l / g pv plasma and 1  :  200 in mice injected with 50  l / g . no circulating pv igg were detected in mice injected with pa - treated plasma . in all of the wt mice , igg deposits were observed with a minimal dose of 30  l / g pv plasma ( 177  g / g pv igg ) . at this dose , about 10% of mice displayed clinical evidence of disease . with an administered dose of 50  l / g pv plasma ( 295  g / g pv igg ) , 75% of the wt mice developed blisters . when a dose - response study was performed on cd8/ mice , a lower incidence of pemphigus was observed . in particular , with a dose of 30  l / g pv plasma , 0% ( 0/5 ) of the cd8/ mice injected showed evidence of disease as compared with 9.5% ( 4/42 ) of c57bl/6 mice ( difference statistically not significant ) . with an injected dose of 50  l / g , 44% ( 13/29 ) of the cd8/ mice developed pv lesions , compared with 72% ( 26/36 ) of the control group ( difference statistically significant ) ( figure 2 ) . the immunopathogenesis of pv involves both humoral and cell - mediated response . while antibodies to desmoglein are pathogenic , cd4 + , cd8 + , and nk cells it is known that cd4 + th1 cells promote the igg1 production by means of b cells , while igg4 , iga , and ige autoabs are induced by the cooperation of cd4 + th2 with b cells . the function of cd8 + cells in the pathogenesis of pv is still unclear , but their role has been hypothesized by some authors . they occasionally observed that cd8 + t cells from patients with active pv were responsive to in vitro stimulation with dsg 3 . specifically they secreted il-2 and inf- but not il-4 or il-5 , being the first cytokines implicated in the cell - mediated immune response and the latters in the humoral one . this suggests a role for cd8 + t cells in the cell - mediated pathogenesis of pv . the characterization of the infiltrate of pv human skin lesions showed mostly cd4 + t - cells and less commonly , about 10% , cd8 + cells . in our study we analyzed the t cell infiltrate of skin lesions in patients affected by pv by means of immunohistochemistry for the expression of cd3 , cd4 , and cd8 . the results confirm the data of the literature because cd4 + are more expressed in pv human skin lesions than cd8 + with a ratio of 2.7 . control mice ( wt ) developed pv lesions ( 9.5% with 30  l / g and 72% with 50  l / g ) , whereas cd8 knockout group was relatively resistant ( 0% with 30  l / g and 44% with 50  l / g ) . as expected , mice that received 50  l / g pv plasma developed more pv lesions than those injected with 30  l / g . histological analyses and dif and iif tests confirmed the diagnosis of pv in mice that presented cutaneous lesions after the injection of pv plasma , while in the totality of mice treated with igg deprived plasma , all tests were negative ( figure 1 ) . these results suggest that cd8 + t cells may play a role in the pathogenesis of pv . cd8 + t cells mediate immunity in part through granzymes or inducing apoptosis by the fas / fas ligand ( fasl ) system . this death cell pathway depends mainly on fas / fasl interaction through the activation of the caspases system . some reports showed that , in keratinocytes treated with pemphigus sera , the activation of caspases was observed . an in vitro study showed that the addiction of anti - fasl abs has partially inhibited the igg - pv induced apoptosis in cultures of keratinocytes . we thus suggest a role of cd8 + t lymphocytes in the pathogenesis of pv which in part may be mediated through perhaps fas - fasl signaling .
pemphigus vulgaris ( pv ) is an autoimmune blistering disease whose pathogenesis involves both humoral and cell - mediated immune response . though the pathogenetic role of autoantibodies directed against desmoglein 3 is certain , a number of other factors 
 have been suggested to determine acantholysis in pv . in this study 
 we examined the possible role of cd8 + t cells in the development of acantholysis by a passive transfer of pv autoantibodies using cd8 deficient mice , and we also studied the inflammatory infiltrate of pv skin lesions by immunohistochemical staining . 
 the results of the immunohistochemical staining to study the expression of cd3 , cd4 , and cd8 in pv skin lesions showed that cd4 + are more expressed than cd8 + in the inflammatory infiltrate of pv lesions , confirming the data of the previous literature . 
 the passive transfer study showed a lower incidence of pemphigus in the group of cd8 deficient mice compared to the control one of wild - type mice . 
 these results suggest that cd8 + t cells may play a role in the pathogenesis of pv , perhaps through the fas / fasl pathway .