research article a possible role for cd8+ t lymphocytes in

Hindawi Publishing CorporationMediators of InflammationVolume 2013 Article ID 764290 5 pageshttpdxdoiorg1011552013764290

Research ArticleA Possible Role for CD8+ T Lymphocytes in the Cell-MediatedPathogenesis of Pemphigus Vulgaris

Federica Giurdanella1 Luca Fania1 Maria Gnarra1 Paola Toto2 Daniela Di Rollo3

Daniel N Sauder45 and Claudio Feliciani1

1 Department of Dermatology Policlinico A Gemelli Hospital Catholic University of the Sacred HeartLargo Agostino Gemelli 8 00168 Rome Italy

2 Department of Dermatology University G DrsquoAnnunzio of Chieti-Pescara Via dei Vestini 5 66013 Chieti Italy3 Department of Medicine and Aging Science University G DrsquoAnnunzio of Chieti-Pescara Via dei Vestini 31 66013 Chieti Italy4Department of Dermatology Princeton University Hospital 253 Witherspoon Street Princeton NJ 08540 USA5 Faculty of Medicine University of Ottawa 451 Smyth Rd Ottawa ON Canada K1H8M5

Correspondence should be addressed to Claudio Feliciani felicianirmunicattit

Received 29 July 2013 Accepted 11 October 2013

Academic Editor Dennis Daniel Taub

Copyright copy 2013 Federica Giurdanella et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Pemphigus vulgaris (PV) is an autoimmune blistering disease whose pathogenesis involves both humoral and cell-mediatedimmune response Though the pathogenetic role of autoantibodies directed against desmoglein 3 is certain a number of otherfactors have been suggested to determine acantholysis in PV In this study we examined the possible role of CD8+ T cells inthe development of acantholysis by a passive transfer of PV autoantibodies using CD8 deficient mice and we also studied theinflammatory infiltrate of PV skin lesions by immunohistochemical staining The results of the immunohistochemical stainingto study the expression of CD3 CD4 and CD8 in PV skin lesions showed that CD4+ are more expressed than CD8+ in theinflammatory infiltrate of PV lesions confirming the data of the previous literature The passive transfer study showed a lowerincidence of pemphigus in the group of CD8 deficient mice compared to the control one of wild-type mice These results suggestthat CD8+ T cells may play a role in the pathogenesis of PV perhaps through the FasFasL pathway

1 Introduction

Pemphigus vulgaris (PV) is a life-threatening autoimmuneblistering disease mediated by autoantibodies (autoAbs)directed against desmogleins (Dsg) located on the surface ofkeratinocyte cells (KC) This leads to an intraepithelial lossof adhesion called acantholysis and clinically it presents withvescicles and blisters [1] AutoAbs in PV are directed mainlyagainst desmoglein 3 (Dsg 3) a desmosomal glycoproteinsituated in the skin predominantly in the suprabasilar epi-dermal layer and less frequently against desmoglein 1 [2]Though the pathogenetic role of antidesmoglein autoAbs iscertain the exact mechanism through which they lead toacantholysis is still incompletely understood Complement[3] plasminogen-plasmin [4] cytokines [5] cell-mediatedimmunity and other autoantibodies such as anticholinergicreceptor antibodies have been suggested in determining

acantholysis in PV [6] Studies conducted so far regarding therole of T cells involved mainly CD4+ lymphocytes for theircooperation with B cells and subsequently for the inductionand regulation of autoAbs production [7] The function ofCD8+ T cells has not been explored yet but some authorshypothesize their role in cell-mediated pathogenesis of PV[8] Other studies suggested a possible role of natural killer(NK) cells [9] as well as Fas and caspase 8 in PV [10]These moleculesrsquo function in the apoptosis mechanism iswell known In PV these molecules result in a shrinking ofkeratinocytes that leads to detachment inducing acantholysis[11] Fas is a member of the tumor necrosis factor (TNF)receptor family that is bound by Fas ligand expressed on TCD8+ cells In this study we sought to evaluate the role ofCD8+ cells performing a passive transfer of PV autoAbs usingCD8 deficient mice (CD8minusminus) The results of these studiessuggest a role for CD8 in the pathogenesis of PV

2 Mediators of Inflammation

2 Materials and Methods

21 Immunohistochemistry Immunohistochemical stainingwas performed using the alkaline phosphatase-antialkalinephosphatase (APAAP) method on 7 120583m skin sections of 7PV patients [12] Monoclonal antibodies against CD3 (1 20DAKO Glostrup Denmark) CD4 (1 20 DAKO) and CD8(1 20 DAKO) were used For the quantitative study stainedcells were first counted in three consecutive microscopicfields (250x) both in the dermis and in the epidermis andthen summed the average value was then calculated

22 Preparation of Pemphigus IgG Plasmawas obtained fromthe plasmapheresis of one patient with clinical histologicand immunologic features consisting of the diagnosis of PVduring the acute phase of the disease Total IgG concentrationwas measured by nephelometry using monospecific goatanti-human IgG (Beckman Instruments Missasauga ONCanada) Pemphigus Ab titers were measured by indirectimmunofluorescence (IIF) using monkey esophagus epithe-lium as the tissue substrate [13] As a negative control IgGfractions were isolated and removed from PV plasma usingprotein A (PA) Isolation of IgG fractions from PV plasmawas achieved by standardized technique using staphylococcalprotein A coupled to Sepharose 4B [14] (Pharmacia BiotechUppsala Sweden) PA was washed four times in cold PBSand finally incubated with PV plasma overnight at 4∘CThe supernatant was collected and used as negative controlAbsence of IgG fractions in the control plasma was assessedby IIF staining on a monkey esophagus epithelium substrateand confirmed by nephelometry PV plasma and controlplasma were filter sterilized with Millex (pore size 022mmMillipore Bedford MA) and stored at minus20∘C

23 Mice The following strains were used CD8minusminus andC57BL6 (CD8minusminus control) All mice were housed and bredunder specific pathogen-free conditions in the animal facilityof the Sunnybrook Health Science Centre Neonates (lt24 hof age) were used An average of 15 mice within eachexperimental group was used and each experiment wasrepeated at least three times All animal procedures wereapproved by the Sunnybrook Health Science Centre animalcare committee

CD8minusminus Mice The generation of mice homozygous for CD8gene mutations (CD8minusminus) was obtained by disruption ofthe Lyt-2 gene through homologous recombination and themutation was interbred into the C57BL6 background beforegenerating CD8-deficient (CD8minusminus) mice Mice homozygousfor the defect were used as the knockout (KO) mice with thewild-type (WT) animals serving as the nondeficient controls

24 Passive TransferModel To induce PV inmice we utilizedthe model of Anhalt et al [15] with minor modificationsBriefly plasma was injected intradermally in the dorsal areainto neonatal mice through a 30-gauge needleThe total doseadministered ranged from 30 to 50120583Lg of body weight ina single administration We chose a dose of 30 120583Lg becausethis was the minimum dose inducing the disease in WT

mice CD8minusminus mice and C57BL6 mice were injected withthe same dose of PV plasma As a negative control genetargeted mutant mice and WT mice were injected withplasma depleted of IgG by treatment with protein A Micewere examined 24 h after the injections Cutaneous lesionsconsisting of intact blisters or erosions were enumerated

25 Tissue Specimens Staining Lesional and perilesional skinwere obtained for light microscopy and direct immunofluo-rescence (DIF) 24 h after injection with PV IgG At the timeof biopsies serum was also obtained and assayed for IIF on amonkey esophagus epithelium to detect the IgG titer

26 Direct Immunofluorescence Perilesional skin was biop-sied and specimens were snap frozen in liquid nitrogen untiluse Cryostat sections (5120583m)were used andDIF studies wereperformed according to standard techniques [16] Brieflyspecimens were washed in PBS for 10 minutes incubated for30 minutes with FITC-conjugated F(ab1015840)

2

fragment of rabbitanti-human IgG specific for 120574-chains (1 25 Dako GlostrupDenmark) and washed in PBS for 15 minutes Slides werecovered with buffered glycerol and results were read in aNikon Optiphot immunofluorescence microscope (NikonMelville NY)

27 Indirect Immunofluorescence Sera were collected 24 hafter PV IgG treatment or PA treatment (IgG depleted) andIIF studies were performed according to standard techniques[16] Cryostat sections (5 120583m) of monkey esophagus wereused as substrate washed for 10min in PBS incubated for30min with different concentrations of sera (1 1-1 600)washed in PBS for 15min labeled with FITC-conjugatedF(ab1015840)

2

fragment of rabbit anti-human IgG (DAKOGlostrupDenmark) for 30min and then washed again in PBS for10min Slides were coveredwith buffered glycerol and resultswere examined using a NikonOptiphot immunofluorescencemicroscope (Nikon Melville NY)

28 Histologic Technique Skin biopsies frommice were fixedin 10 formalin and stained with hematoxylin and eosin

29 Statistical Analysis Data regarding the incidence of thedisease in KO and control mice were analyzed using the 1205942test a 119875 value lt005 was considered to be significant

3 Results

31 Immunohistochemistry CD3-CD4-CD8 CD3+ T cellswere detected both in the dermis (328 plusmn 16 cells counted asmentioned in Section 2) with a perivascular distribution andin the epidermis (79 plusmn 28) of all patients CD4+ T cells werefound in the superficial and papillary dermis (336plusmn48) withscattered and perivascular distribution and a fewer numberof them were detected in the basal and suprabasal layers nearthe dermal-epidermal junction (42plusmn12) A number of CD8+T cells (142 plusmn 16) were observed in perivascular areas ofdermal lesional skin (CD4CD8 = 27) (Table 1)

Mediators of Inflammation 3

Table 1 T cellular markers identified in human skin lesions of PVpatients by immunohistochemistry The calculated average numberof stained cells in three consecutive microscopic fields (250x) isreported

Dermis EpidermisCD3 328 plusmn 16 79 plusmn 28

CD4 336 plusmn 48 42 plusmn 12


32 Pemphigus Vulgaris IgG Pemphigus plasma wasobtained as described in Section 2 IIF for PV IgG usingmon-key esophagus as substrate demonstrated a titer of 1 2460and an IgG concentration of 59mgmL was measured bynephelometry PA-treated plasma showed absence of inter-cellular staining on monkey esophagus and IgG levels werebelow the level of detection using the nephelometric analysis

33 Passive Transfer of PV The passive transfer of PV IgGdemonstrated a direct correlation between the amount of PVIgG injected and the incidence of the disease Acantholysisand inflammatory infiltrate were evident in mice givenplasma containing PV IgG and absent in mice injected withIgG-depleted plasmaThe epidermis ofmice injectedwith PVIgG who developed PV lesions showed human IgG bound tothe intercellular cell surface by DIF No staining was foundin mice injected with PA-treated plasma No difference wasobserved in the intensity of fluorescence in different strainsof mice treated with an equal dose of PV IgGThe IgG titer inall PV plasma injected mice detected by IIF ranged between1 100 in mice treated with 30 120583Lg PV plasma and 1 200in mice injected with 50 120583Lg No circulating PV IgG weredetected inmice injected with PA-treated plasma In all of theWT mice IgG deposits were observed with a minimal doseof 30 120583Lg PV plasma (177 120583gg PV IgG) At this dose about10 of mice displayed clinical evidence of disease With anadministered dose of 50120583Lg PV plasma (295120583gg PV IgG)75 of the WT mice developed blisters

When a dose-response study was performed on CD8minusminusmice a lower incidence of pemphigus was observed Inparticular with a dose of 30120583Lg PV plasma 0 (05) ofthe CD8minusminus mice injected showed evidence of disease ascompared with 95 (442) of C57BL6 mice (differencestatistically not significant)With an injected dose of 50120583Lg44 (1329) of the CD8minusminus mice developed PV lesionscompared with 72 (2636) of the control group (differencestatistically significant) (Figure 2)

4 Discussion and Conclusions

The immunopathogenesis of PV involves both humoral andcell-mediated response While antibodies to desmoglein arepathogenic CD4+ CD8+ and NK cells are also implicatedThe function of CD4+ cells has previously been examined Itis known that CD4+ Th1 cells promote the IgG1 productionby means of B cells while IgG4 IgA and IgE autoAbs areinduced by the cooperation of CD4+ Th2 with B cells [9]The function of CD8+ cells in the pathogenesis of PV is

(a)

(b)

(c)

(d)

Figure 1 (a)Histology of a PV lesion from awild-typemice injectedwith PV IgG (b) Direct immunofluorescence in wild-type miceinjected with PV IgG showing IgG bounded to the intercellularcell surface (100x) (c) Histology of mice treated with IgG deprivedplasma no acantholysis is observed Immunofluorescence studieswere also negative (data not shown) (d) A large erosion on the backof a mice treated with PV IgG


0

10

20

30

40

50

60

70

80

30

(120583Lg)

()

C57BL6CD8 KO

50

Figure 2 Incidence of disease in KO mice and control-C57BL6mice in passive transfer model of IgG PV With a dose of 30120583LgPV plasma 0 (05) of the CD8minusminus mice developed PV comparedwith 95 (442) of C57BL6miceWith an injected dose of 50120583Lg44 (1329) of the CD8minusminus mice showed PV lesions compared with72 (2636) of the control group

still unclear but their role has been hypothesized by someauthors They occasionally observed that CD8+ T cells frompatientswith active PVwere responsive to in vitro stimulationwith Dsg 3 Specifically they secreted IL-2 and INF-120574 but notIL-4 or IL-5 being the first cytokines implicated in the cell-mediated immune response and the latters in the humoraloneThis suggests a role for CD8+ T cells in the cell-mediatedpathogenesis of PV [9]

The characterization of the infiltrate of PV human skinlesions showed mostly CD4+ T-cells and less commonlyabout 10CD8+ cells [17] In our studywe analyzed theT cellinfiltrate of skin lesions in patients affected by PV bymeans ofimmunohistochemistry for the expression of CD3 CD4 andCD8 The results confirm the data of the literature becauseCD4+ are more expressed in PV human skin lesions thanCD8+ with a ratio of 27 Control mice (WT) developed PVlesions (95 with 30120583Lg and 72 with 50 120583Lg) whereasCD8knockout groupwas relatively resistant (0with 30 120583Lgand 44 with 50 120583Lg) As expected mice that received50120583Lg PV plasma developed more PV lesions than thoseinjected with 30 120583Lg Histological analyses and DIF and IIFtests confirmed the diagnosis of PV in mice that presentedcutaneous lesions after the injection of PV plasma while inthe totality of mice treated with IgG deprived plasma all testswere negative (Figure 1) These results suggest that CD8+ Tcells may play a role in the pathogenesis of PV CD8+ T cellsmediate immunity in part through granzymes or inducingapoptosis by the FasFas ligand (FasL) systemThis death cellpathway dependsmainly on FasFasL interaction through theactivation of the caspases system Some reports showed thatin keratinocytes treated with pemphigus sera the activationof caspases was observed Other reports demonstrated that

inhibitors of caspases provoke the blockade of acantholysis[11] An in vitro study showed that the addiction of anti-FasLAbs has partially inhibited the IgG-PV induced apoptosis incultures of keratinocytes [11]

We thus suggest a role of CD8+ T lymphocytes in thepathogenesis of PV which in part may be mediated throughperhaps Fas-FasL signaling

References

[1] J R Stanley M Yaar P Hawley-Nelson and S I KatzldquoPemphigus antibodies identify a cell surface glycoprotein syn-thesized by human andmouse keratinocytesrdquo Journal of ClinicalInvestigation vol 70 no 2 pp 281ndash288 1982

[2] M Amagai ldquoPemphigus autoimmunity to epidermal cell adhe-sion moleculesrdquo Advances in Dermatology vol 11 pp 319ndash3531996

[3] S Kawana W D Geoghegan R E Jordon and S NishiyamaldquoDeposition of the membrane attack complex of complementin pemphigus vulgaris and pemphigus foliaceus skinrdquo Journalof Investigative Dermatology vol 92 no 4 pp 588ndash592 1989

[4] P Fabbri T Lotti and E Panconesi ldquoPathogenesis of pemphi-gusThe role of epidermal plasminogen activators in acantholy-sisrdquo International Journal of Dermatology vol 24 no 7 pp 422ndash425 1985

[5] S A Grando B T Glukhenky G N Drannik E V Epshtein AP Kostromin and T A Korostash ldquoMediators of inflammationin blister fluids from patients with pemphigus vulgaris andbullous pemphigoidrdquo Archives of Dermatology vol 125 no 7pp 925ndash930 1989

[6] R S Kalish ldquoPemphigus vulgaris the other half of the storyrdquoJournal of Clinical Investigation vol 106 no 12 pp 1433ndash14352000

[7] K Nishifuji M Amagai M Kuwana T Iwasaki and TNishikawa ldquoDetection of antigen-specific B cells in patientswith pemphigus vulgaris by enzyme-linked immunospot assayrequirement of T cell collaboration for autoantibody produc-tionrdquo Journal of Investigative Dermatology vol 114 no 1 pp 88ndash94 2000

[8] M Hertl and C Veldman ldquoT-cellular autoimmunity againstdesmogleins in pemphigus an autoantibody-mediated bullousdisorder of the skinrdquo Autoimmunity Reviews vol 2 no 5 pp278ndash283 2003

[9] H Takahashi M Amagai A Tanikawa et al ldquoT helper type 2-biased natural killer cell phenotype in patients with pemphigusvulgarisrdquo Journal of Investigative Dermatology vol 127 no 2 pp324ndash330 2007

[10] M Puviani A Marconi E Cozzani and C Pincelli ldquoFas ligandin pemphigus sera induces keratinocyte apoptosis through theactivation of caspase-8rdquo Journal of Investigative Dermatologyvol 120 no 1 pp 164ndash167 2003

[11] S A Grando J-C Bystryn A I Chernyavsky et al ldquoApop-tolysis a novel mechanism of skin blistering in pemphigusvulgaris linking the apoptotic pathways to basal cell shrinkageand suprabasal acantholysisrdquo Experimental Dermatology vol18 no 9 pp 764ndash770 2009

[12] J L cordell B Falini and W N Erber ldquoImmunoenzymaticlabeling of monoclonal antibodies using immune complexesof alkaline phosphatase and monoclonal anti-alkaline phos-phatase (APAAP complexes)rdquo Journal of Histochemistry andCytochemistry vol 32 no 2 pp 219ndash229 1984


[13] C Feibelman G Stolzner and T T Provost ldquoPemphigusvulgaris Superior sensitivity of monkey esophagus in the deter-mination of pemphigus antibodyrdquoArchives of Dermatology vol117 no 9 pp 561ndash562 1981

[14] T JMiller andHO Stone ldquoThe rapid isolation of ribonuclease-free immunoglobulin G by protein A-sepharose affinity chro-matographyrdquo Journal of Immunological Methods vol 24 no 1-2pp 111ndash125 1978

[15] G J Anhalt R S Labib and J J Voorhees ldquoInduction of pem-phigus in neonatal mice by passive transfer of IgG from patientswith the diseaserdquoTheNewEngland Journal ofMedicine vol 306no 20 pp 1189ndash1196 1982

[16] G Pohla-Gubo and H Hintner ldquoDirect and indirect immuno-fluorescence for the diagnosis of bullous autoimmune diseasesrdquoDermatologic Clinics vol 29 no 3 pp 365ndash372 2011

[17] J Schaller H W Niedecken E Biwer J A Stefan H WKreysel and U F Haustein ldquoCharacterization of lymphocyticinfiltrate cells in bullous pemphigoid and pemphigus vulgarisrdquoDermatologische Monatsschrift vol 176 no 11 pp 661ndash6681990

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2 Materials and Methods

21 Immunohistochemistry Immunohistochemical stainingwas performed using the alkaline phosphatase-antialkalinephosphatase (APAAP) method on 7 120583m skin sections of 7PV patients [12] Monoclonal antibodies against CD3 (1 20DAKO Glostrup Denmark) CD4 (1 20 DAKO) and CD8(1 20 DAKO) were used For the quantitative study stainedcells were first counted in three consecutive microscopicfields (250x) both in the dermis and in the epidermis andthen summed the average value was then calculated

22 Preparation of Pemphigus IgG Plasmawas obtained fromthe plasmapheresis of one patient with clinical histologicand immunologic features consisting of the diagnosis of PVduring the acute phase of the disease Total IgG concentrationwas measured by nephelometry using monospecific goatanti-human IgG (Beckman Instruments Missasauga ONCanada) Pemphigus Ab titers were measured by indirectimmunofluorescence (IIF) using monkey esophagus epithe-lium as the tissue substrate [13] As a negative control IgGfractions were isolated and removed from PV plasma usingprotein A (PA) Isolation of IgG fractions from PV plasmawas achieved by standardized technique using staphylococcalprotein A coupled to Sepharose 4B [14] (Pharmacia BiotechUppsala Sweden) PA was washed four times in cold PBSand finally incubated with PV plasma overnight at 4∘CThe supernatant was collected and used as negative controlAbsence of IgG fractions in the control plasma was assessedby IIF staining on a monkey esophagus epithelium substrateand confirmed by nephelometry PV plasma and controlplasma were filter sterilized with Millex (pore size 022mmMillipore Bedford MA) and stored at minus20∘C

23 Mice The following strains were used CD8minusminus andC57BL6 (CD8minusminus control) All mice were housed and bredunder specific pathogen-free conditions in the animal facilityof the Sunnybrook Health Science Centre Neonates (lt24 hof age) were used An average of 15 mice within eachexperimental group was used and each experiment wasrepeated at least three times All animal procedures wereapproved by the Sunnybrook Health Science Centre animalcare committee

CD8minusminus Mice The generation of mice homozygous for CD8gene mutations (CD8minusminus) was obtained by disruption ofthe Lyt-2 gene through homologous recombination and themutation was interbred into the C57BL6 background beforegenerating CD8-deficient (CD8minusminus) mice Mice homozygousfor the defect were used as the knockout (KO) mice with thewild-type (WT) animals serving as the nondeficient controls

24 Passive TransferModel To induce PV inmice we utilizedthe model of Anhalt et al [15] with minor modificationsBriefly plasma was injected intradermally in the dorsal areainto neonatal mice through a 30-gauge needleThe total doseadministered ranged from 30 to 50120583Lg of body weight ina single administration We chose a dose of 30 120583Lg becausethis was the minimum dose inducing the disease in WT

mice CD8minusminus mice and C57BL6 mice were injected withthe same dose of PV plasma As a negative control genetargeted mutant mice and WT mice were injected withplasma depleted of IgG by treatment with protein A Micewere examined 24 h after the injections Cutaneous lesionsconsisting of intact blisters or erosions were enumerated

25 Tissue Specimens Staining Lesional and perilesional skinwere obtained for light microscopy and direct immunofluo-rescence (DIF) 24 h after injection with PV IgG At the timeof biopsies serum was also obtained and assayed for IIF on amonkey esophagus epithelium to detect the IgG titer

26 Direct Immunofluorescence Perilesional skin was biop-sied and specimens were snap frozen in liquid nitrogen untiluse Cryostat sections (5120583m)were used andDIF studies wereperformed according to standard techniques [16] Brieflyspecimens were washed in PBS for 10 minutes incubated for30 minutes with FITC-conjugated F(ab1015840)

2

fragment of rabbitanti-human IgG specific for 120574-chains (1 25 Dako GlostrupDenmark) and washed in PBS for 15 minutes Slides werecovered with buffered glycerol and results were read in aNikon Optiphot immunofluorescence microscope (NikonMelville NY)

27 Indirect Immunofluorescence Sera were collected 24 hafter PV IgG treatment or PA treatment (IgG depleted) andIIF studies were performed according to standard techniques[16] Cryostat sections (5 120583m) of monkey esophagus wereused as substrate washed for 10min in PBS incubated for30min with different concentrations of sera (1 1-1 600)washed in PBS for 15min labeled with FITC-conjugatedF(ab1015840)

2

fragment of rabbit anti-human IgG (DAKOGlostrupDenmark) for 30min and then washed again in PBS for10min Slides were coveredwith buffered glycerol and resultswere examined using a NikonOptiphot immunofluorescencemicroscope (Nikon Melville NY)

28 Histologic Technique Skin biopsies frommice were fixedin 10 formalin and stained with hematoxylin and eosin

29 Statistical Analysis Data regarding the incidence of thedisease in KO and control mice were analyzed using the 1205942test a 119875 value lt005 was considered to be significant

3 Results

31 Immunohistochemistry CD3-CD4-CD8 CD3+ T cellswere detected both in the dermis (328 plusmn 16 cells counted asmentioned in Section 2) with a perivascular distribution andin the epidermis (79 plusmn 28) of all patients CD4+ T cells werefound in the superficial and papillary dermis (336plusmn48) withscattered and perivascular distribution and a fewer numberof them were detected in the basal and suprabasal layers nearthe dermal-epidermal junction (42plusmn12) A number of CD8+T cells (142 plusmn 16) were observed in perivascular areas ofdermal lesional skin (CD4CD8 = 27) (Table 1)











(a)

(b)

(c)

(d)



0

10

20

30

40

50

60

70

80

30

(120583Lg)

()

C57BL6CD8 KO

50






References
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























(a)

(b)

(c)

(d)



0

10

20

30

40

50

60

70

80

30

(120583Lg)

()

C57BL6CD8 KO

50






References
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

10

20

30

40

50

60

70

80

30

(120583Lg)

()

C57BL6CD8 KO

50






References
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article a possible role for cd8+ t lymphocytes in

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