HL Α Typing:
Methodology
and
Clinical Aspects
Volume II
Editors
Soldano Ferrone, M.D., Ph.D.
Professor
Department of Pathology
College of Physicians and Surgeons
Columbia University
New York, New York
Bjarte G. Solheim, M.D., Ph.D.
Head, Blood Typing Laboratory
Rikshospitalet, The National Hospital
Oslo, Norway
Olli
•
CRC Press
Boca Raton,
•i
, Inc.
Florida
105
Chapter 10
HLA-Α,Β R E S T R I C T I O N O F C Y T O T O X I C Τ C E L L S
E. Goulmy
T A B L E O F C O N T E N T S
I. Background and Models 106
A. Backgrounds 106
B. Models 107
1. HLA Restricted Anti-H-Y Cytotoxicity 107
2. HLA Restricted Anti-Virus Cytotoxicity 107
a. Epstein-Barr Virus 107
b. Influenza Virus 107
c. Measles Virus 108
d. Herpes Simplex Virus and Human Cytomegalo
Virus 108
3. HLA Restriction of Chemically Modified Cells 108
a. Tnnitrophenyl 108
b. Dinitrofluorbenzene 109
c. Fluorescein Isothiocyanate 109
Π. Technical Aspects 109
A. Cell Mediated Lympholysis Assay 109
1. Introduction or Sensitization Phase 109
a. Preparation of the Effector Cells 109
b. Preparation of the Target Cells 109
2. Destructive or Effector Phase 109
a. Preparation of the Effector Cells 109
b. Preparation of the
5 1Cr Labeled Target Cells 109
c. Assay 110
3. Harvesting and Counting 110
4. Calculation and Interpretation 110
B. Monolayer Absorption Techniques 111
1. Preparation of the Monolayers 112
2. Results and Interpretation of Anti-H-Y Killer Cell Activity . . . 112
a. Depletion in Cytotoxicity of the HLA-A2 Restricted Anti
H-Y Cytotoxic Τ Cells 112
b. Reduction in Cytotoxicity of the HLA-A2 and HLA-B7
Restricted Anti H-Y Cytotoxic Τ Cells 112
C. Target Cell Inhibition Studies 114
1. Cold Target Inhibition of the HLA-A2 Restricted Anti H-Y
Cytotoxic Cells 114
2. Cold Target Inhibition of the HLA-A2 and HLA-B7 Restricted
Anti H-Y Cytotoxic Cells 114
D. Long-Term Culture and Cloning Procedures 115
1. Long-Term Growth of the HLA-A2 Restricted Anti H-Y
Cytotoxic Τ Cell Lines 115
2. Cloning 116
3. Implications of Long-Term Cultures and Cloning Techniques 118
• ' \ •• - · •'"' • * * - • * * *
106 HLA Typing Mcthodology <χηά Clmiial Aspccis
III Relevante in Chnieal Meditme
Aeknowledgments
Rcfercnces
118
119
119
I BACKGROUNDS AND MODELS Α Backgrounds
The availabihty of an in vitro technique for the induction and differentation of spe cific cytolytic effector Τ eells has made d significant contribution to the understanding of the role of the Major Histocompatibihty Complex (MHC) antigens in the immune response, and provided as in vitro modet of the homograft reaction Hirschhorn and co workers1 were the first to demonstrate thal human penpheral blood lymphocytes
(PBL) (.uitiired with human fibroblasts from unrelated individuals were able to lyse allogeneic fibroblasts Hayry and Defendi2 desenbed that mouse lymphocytes sensi
ti?ed in vitro with allogeneic lymphocytes were able to destroy only the lymphoma Iine target cells which carned the specific sensiti/ed antigens and did not affect cells which were isogeneic to the original responding cells In man, Solhday and Bach1 showcd
preferential destruction of lymphoblastoid cell hnes isogeneic to the sensitmng cell, although cross reactivity with other cell hnes was observed The so called Cell Me diated Lympholysis (CMI ) technique was subsequently developcd by I ightbody et al * The technique consists of two in vitro phases an induction phasc in which lympholytic effector cells are indueed, and an effector phasc in which the effector cells lyse chro mium labeled target cells The generation of effector cells in the first step is necessary in order to obtain measurable cytotoxicity in the second step
Α direet CMI , so called LMC (l ymphocyte Mediated Cytolysis), consists of only one in vitro step PBLs, removed from, foi example, an in vivo sensitized patient are tested without an induction phase directly against chrommm labeled target cells
The CML technique was onginally used to study the genetics of the MHC The determinants recogmzcd by that technique are in all probabihty class I (HLA Α Β C) specificitics11 7 but also class II (HLA D) antigens (Mawas et al H I eigh^ry et al 9
and Albrcchtsen et al ' ) or determinants closely hnked lo them They have also been reierred to as CD determinants or cytotoxic defincd determinants
/inkcrnagcl and Doherty" used the CML lechnique to study the specific uiteraction ot virus and MHC determinants which has been of major importance for our under standmg of the immune response in general and the role of the MHC determinants in particular The observed phenomenon was called MHC restnction and was first stud icd by them in the mouse in a model of virus indueed lymphocytic chonomenmgitis soon followcd by a niodcl of hapten MHC restnction (Shearer")
The 11 2 rcstnclion phenomenon was not hmited only to virus and chemically aliered cell surface produets Bevan12 demonstrated Η 2 restneted cytotoxic Τ teils directed
towards non Η 2 minor histocompatibihty antigens Gordon et al '3 showed the in
voivement of the Η 2 rcgion produets to obtain eytotoxie Τ cell resporse against the male specific anligcn, Η Υ
1 he mechunism by which theie Η 2 restneted eytotoxie Γ cells recognize modified
107 Single recogmtion model) have been proposed 4 More recently expenments by Bur akoff et al " may suggesl that cytotoxic Τ cell responses against alloantigens may be compatible with responses against autologous MHC products modified for example by viruses
Β Models
/ HLA Restneted Anti Η Υ Cytotoxicity
The involvement of the MHC in Τ cell mediated HLA restricted cytotoxic rcsponse against foreign in rodents was confirmed in man and has been detected for ι minor histocompatibihty antigen natnely the male specific antigen Η Υ (Goulmy et al ) '' In this example the pumary sensitization occurred in vivo PBL of a bone mnrrow transplanted female aplastic anemia patienl showed direct and mdircu cell mediated cytotoxicity only against male target cells which carncd one of the HLA anti^ens ol the original sensitizing cell namely the HLA A2 antigen This antigen was also present on the patient s lymphocytes
This observation of HLA Α locus produet restnction of cytotoxic Τ cells directed against the minor histocompatibihty antigen Η Υ was later confirmed in four other cases (all multitransfused female aplastic anemia patients) The subsequent cases dem onstrate also that the samc magmtude of Η Υ immune cytotoxicity could be obseived in conjunction with HLA determinants coded for by diflerent haplotypes (Goulmy et il ') Experiments by Singal et al "" have confirmed the killing of HLA A2 positive male target cells by in vivo immunized HLA A2 positive females They showcd that pregnancy in ltself is sufficienl to induce sensitization to HI Α restneted cytoloxicity
2 HLA Restncled Anti Virus Cytotoxicity
a Epstem Barr Virus (EBV)
Tursz et al lft have suggested that HLA Α and Β region determinants are necessary for anti viral Τ cell mediated cytotoxicity towards ÜBV sensitized Τ cells They re ported that EBV sensitized penpheral Τ lymphocytes from patients with infcctious mononucleosis sensitized against EBV failed to lyse EBV infected Daudi cells which apparently lack the HLA class I antigens at the membrane but could lyse all other LBV cell lines without any apparent HLA specificity On the other hand no evidence for allogeneic restnction in this system was apparent *• Lipinski et al * suggested that the HLA region appears to act at two different leveis in the Τ cell mediated lysis of EBV infected cells Experiments by Rickinson et al 9 confirmed that the HLA Α and Β region products indeed play a role in the Τ cell recogmtion of CBV infected Β cells Λ recent report by Misko et al 9u strongly indicated that Τ cell cytotoxicity to TBV is restneted by HLA antigens In this (in vitro) »tudy 14 days eultures of lympho cytes from EBV seropositive donors ha\e been investigated and specificity was dem onstrattd using FBV infected lymphoblastoid celi lines as target cells
b Influenza Virus
McMichael and Ting 2° who showed that cytotoxic Τ lymphocytes and influcn/a virus infected target cells must share a part cular HLA antigen Cytotoxic effector cells specific for one type of influenza virus A/X31 or B/Hong Kong killed the nutologous and HLA matched infected target cells but not HLA mismatched cells HLA B7 seemed to be the required restneting antigen The HLA A2 antigen seemed to be in adequate in the cytotoxic Τ lymphocyte recogmtion of influenza virus McMichael11 and Biddison et al " have confirmed these findings However both studics reported discrepancies between the serologically defined HLA specificites and the determinants which are recognized by the Τ cells in association with the influenza virus The latter authors discussed the possibihty that Τ cells may even discriminatc between the sero
108 HLA Typing: Mcthodology and CHmcal Aspccts
ogically dcfined HLA antigens and tho.se that are not yet serologicaily distinguisha-ble." Furthermore, Shaw and Biddison24 investigated families to study the genetic con-trol of the in vitro Τ cell responses to influcnza virus-infected autologous cells and found responses that were associated with preferential haplotypes. The results of stud-ies using unrelated donors1 5 demonstrated variable Sytic capacity to virus infected seif target antigens. The degree of responsiveness could be explained on responder, stimu-lator, or target level. The peculiar behavior, i.e., different cytotoxic Τ cell responses towards virus-infected target cells sharing the same HLA antigens, could be explained by a genetically controlled regulation System (Biddison and Shaw).15 These studies have been extended, with observations showing preferential recognition of HLA target an-tigens for the different, but closely related, influenza types A/Hong Kong and B/Hong Kong(Shawet al.).1(i
c. Measles Virus
Another example of the HLA restncted cytotoxic Τ celi function was found in as~ sociation with rneasles virus (Kreth et al.).2 7 The iymphocytes of five patients with aculc measles preferentially lysed measles virus-infected target cells which shared the appropriate HLA-A or -B determinants. Apparently no restriction is iound after the acute phase of the disease.
d. Herpes Simplex Virus (HSV) and Human Cytomegalo Virus (HCMV)
Studies by Sethi et al.2 8 demonstrated HLA restriction in the lytic activity towards HSV and HCMV infected skin fibrobiast target cells. The presence of virus-specific cytotoxic Iymphocytes (CTLs) was shown by using long-term cultures denved from periphera! blood Iymphocytes from in vivo sensitized patients. Α recent report by Quinan et al.i B a demonstrated the development of HLA-restricted CTL in vivo in four bone marrow transplant recipients during acute CMV infection. Earlier attcmpts to demonstrate HLA restncted cytotoxic Τ cells during primary infections have failed; Sleelc et al.2 9 rubella virus; Perrm et al.1 0: vaccinia virus; Pernn et al.J'· measies virus.
3. HLA Restriction of Chemicalty Modtfied Cctte a. Trinitrophenyl (TNP)
The first attempt to demonstate the mvolvement of HLA restriction in a hapten modified System was described by Newman ei a l . " Specific cytotoxic Iymphocytes (CTLs) eücited responses to only the TNP-coupled autologous cells. However, therc
was an absoute need for boo.sting aftcr in vitro priming. Friedman et al.31 reporied, in
a more extensive study, that the cytotoxic activity was mediated by Τ cells, and could
be triggered in vitro. Restriction t o T N P modified 'seif but not for unaltered or TNP-modified allogeneic target celis was described. Shaw et al.1 4 t s have demonstrated that cytolytic activjty could be observed not only against TNP-modified autoiogous cells but also against modified allogeneic cells. They aiso observed differences in donors' responses to the same modified allogeneic seif antigens. The presence of the poly-morphic MHC determinants such as the HLA-A and -B iocus antigens was not an absolute requirement for the human cytotoxic Τ ceil responses to TNP-modified target cells. These authors suggested that the anti TNP Τ cell recognition responses could be associated with multiple recognition struetures of the cell surface determinants. Char-mot and Mawas"1 have confirmed Shaw's findings that MHC restriction, in the TNP Model, is not a gcncral phenomenon. In addition, Seidin and Rieh17, Channot and Mawas'* and Friedman et al.I B reported the requirement of HLA-D region produets to trigger specific TNP-cytotoxic Τ cells in their response to TNP-modified autologous target cells.
-109
b Dimtrofluorbenzene (DNFB)
The hapten DNFB has been used to study whether or not lymphocytes from Dim trochlorobenzene (DNCB) sensitized patients were able to produce cytotoxic responses agamst DNFB treatcd autologous and HLA compatible target cells (Dickmeiss et al ") The obscrved cytotoxicity was directed against only DNFB coupled target cells and was seen (in three cases) in conjunction with the HLA-A2 antigen
c Fluorescem Isothwcyanate (FITC)
Friedman and his colleagues40 also uscd FITC as an hapten for the generation of
cytotoxic Τ cells to FITC-conjugated autologous target cells In addition, they dem-onstrated the mabihty of cytotoxic Τ cells specific for FITC to lyse TNP modified autologous target cells, thus indicating the hapten specific altered seif reactivity The role of MHC determinants in this System is still unclear
II TECHNICAL ASPECTS Α Cell Mediated Lympholysis (CML) Assay
Fhc method that is currently used in our laboratory to detect cytotoxic Τ cell re sponses to the minor histocompatibihty antigen (H Y) will be described
/ Inductwn or Servutizatwn Phase
a Preparation of the Effector Cells
Ulood is collected into sterile bottles containing prescrvative free hepann (50 IU/ mi)
The lymphocytes are separated by Ficoll Ispaque gradient oentrifugation
Cell concentration responder cells 1 χ 10* cell/mf, stimulator cells 1 χ 10* cell/mi (inactivated by 2000 rad y Irradiation) Depending on the amount of cell recovery, 50 mi culturc flasks or 2 mi cluster wells are used The culture flasks are stored at a 45° angle dunng the culture penod
Culturc conditions and duration 37°C, 5% CO^, well humidified ineubator, 144 hr Culture medium RPMI 1640, supplemented with 3 mML glutamine 100 IU peni-cillin/mi, 100 μg streptomycin/mi and 20% heat inactivated pooled human AB serum from male donors
Washing fluid Hanks' BSS supplemented with 50 IU pen« iliin/m!
b Preparation of the Target Cells
Cell concentration 5 to 8 χ 10' cells are cultured in 5 to 8 mi culture medium Phytohemagglutinin (PHA) is added for the culture duration of 72 hr
Additional note fresh or 6 day cultured unstimulated lymphocytes can also be used
as target cells although iower percentages of lysis wil1 be obtained in the effector phase
An cxample (with Special regard to the specific Hi_A A2 restneted anli Η Υ lysis) of the use of stimulated and/or unstimulated target cells is shown in Figure 1
2 Datructive orBffettor Phase a Preparation of the Effector Cells
After the 6 day induction phase, the effector cells aie transferred to 50 mi tubes, centrifugated at 350 χ g for 10 min, resuspended in 1 to 2 mi fresh culture medium Viabihty counting is performed with eosin 0 1% Thereafter the cells are brought to the desired concentration (see c)
b Preparation of "Cr-Labeled Target Cells
The target cells are gently collected from the flasks and transferred to 15 mi conical bottomed tubes Centnluged at 150 x g for 10 min The supernatant is decanted and the target cells are resuspended in the remaining 0 4 mi supernatant
110 HLA Typing Methodology jnd C Imical Aspccis
Effector target rot ο
I I O U R I ! Spi.cifiL lyl c t-dp l u l y dgainsl (wo HLA Λ2 positive null, tarnet ceils ( c o m p a r c o n bclwccn blasls anci PBI s) la ff[ Λ Λ2 ό lar fed LCIU (bldsls) Ih H l Λ Λ2 <J largcl c d l s (PBI ·,) 2 j Hl Λ Λ2 d I irf,cl c d l s (blasts) 2b I H Λ Λ2 d I irgel cclls (PBI s) 1 HI Λ Λ2 9 Idr^ct <-dh (bldsls) ]\r U l U b t lysis cxprcssctl in I U 50 la 1 4 2a
2 8 1b 7 ( 2b 50 3 >10()
I abeling 100 μΟ Naj ''CrO4 Amersham CJS 1P 5 mC i/5mi spec acl 100 to
350 mCi/mg
Intubation I hr in a waterbath al 37°C After incubation approximately 4 tni wash mg fluid ις added gently mixed and centnfuged dt 150 χ g for 10 min The pellet is washed twjce and thereafter resuspended in eulture medium
Viability counting is ptrformed with eosin 0 1% The teils art brought lo a concen trationof I x lOVmf
c Assay
Add 0 1 mi effettor teil Suspension and 0 1 ml target teil Suspension to each weil of a round bottomed microtiter plate Prefeiential effettor teil target teil ratios are 50 1 25 1 5 1 All assays are performed in tuplicalc
Mitrotiter plates with targets and effettors are tentnfuged for 2 mm at 150 χ g Ihen ineubated for 4 hr at 37°C
Spontancous rclease 0 1 ml target cell Suspension and 0 I ml tulture mediuri/well (in tnplitate) are ineubated in a mitrotiter plate for 4 hr at 37°C
Maximum release (in tnpheate) 0 1 ml target teil Suspension and 0 1 ni of a /a pomne solution (10 ml RPMI 1640 4 10 drops 7apomne Coulter Flettronits 1 td ) art intubated for 4 hr at room lemperature
3 Hdrvciting and Counung
All microtiter plates are tentnfuged for 5 min at 500 χ g The supernatants are removed witn the Flow supernatant harvester (the titertek System) (The use of this titer tek System is onginally desenbed by Hirschberg et al 4C") The samples are countea
for 1 or 2 mm in a γ counter (Packard 5260)
4 Calculation and Interpretation
The pertent lysis u> talculated using tht following formula
I xpLnmentdl mcan cpm spontaneuus relt-asc mean cpm
111
χ'
0
0 5 1 0 1 5 2 0
sporuancout. releasc i η cp 5 χ 10
I KiUKI 2 Rüanonship belWLcn sponUtuous and maximum rt
Standard errors of thc means of tnplicates has to be less tKan 5% The rcsults are expressed on a scale in which the spontaneous release value is set to 0% and the maxi mum releasc value to !(K)% (see Figure 2) Spontaneous release which is determined in wells wilh assay medium alone, normally amounts to '5 to 25% of thc maximum release
When only one ef feUor to target (Ε/Τ) ι atio is used, h sis percentages equal or below
10% are considered as negative, 11 to 15% as weakly positive, 16 to 40% as positive, and >40% as strongly positive When thc number oi cells for an expenment are lim iled, positive and negative assignments are made on the basis of a 10% specifit Cr release value This entenon is used also for the assignment CML positivity by the huropean CML study group The expression of percentage lysis in Iytic units (LU) is a useful parameter of CML activity LU 50, *"or example, is the number of effector cells χ I0"4 necessary to obtam 50°/o specific lysis of 104 target cells According to this defimtion, a larger number of LUs reflects a weaker response because more effectors ate required t o c a u s e a 5 0 % CML response
Our laboratory participated in a collaborative study to standardize the CML tech nique The results of four CML Workshops and a recommended European Standard CML technique have been reported 41 AA
Β Monolayer Absorption Technique
Α monolayer absorption technique has been used to test whether killer cells directed to seif HLA associated with the minor histocompatibihty antigen (H-Y) were divisiblc into subsets ot killcr Τ cells (Goulmy et al 17)
112 HLA lypmg Methodology änd Chnical Unabsorbed absorbed on mon 1u absorbed on mon 2 absorbed on mon 3 absorbed on mon L
Effector target latio
[ K r U K I 1 A b s o r p t i o n of Ihc Hl Λ Λ2 rcMnclt-d mti Η Υ L>IO
IOXIC clfei-tor teils Monolayer I H L A A 2 vt <S monolayci 2 Mi Α Λ2 - ve 9 m o n o l a y t r 1 HLA A2 ve 9 monolayet 4 Hl Α Α2 I vc d rtic un ibsorbed incl dbs i r l x d C T [ s art. Itsltd on Hl Λ Λ2 posillvt in ife urya teils
Plus lechmque which allows the partition of cell populations was dtvelopcd in our laboratory by Hamillon et al 4 S based on monolayer absorption studics donc by oth
ers 4' "
1 Preparatwn of the MonoUyers
Monolayers werc prepared from fresh PBl s (resuspended in serurn frce mcdium m a concentration of 20 x 10" cells/mi) adhermg to Petn dishes (35 χ 10 mm) which arc treated with a 50 Mg/mi solulion ol Poly L Lysine (PLL mol wt 230,000 Sigm ι Chemical Co St Louis, Mo ) After incubation for 1 hr al room tempcrature tlic nonadherent teils were removed and Ihe cffeclor cells (adjusled lo 10 χ 10 cells/mf in RPMI 1640 wilh 20% pooled human serum) were genlly overlaid on thc washcd monolayer and incubated for I hr at 37°C Thereafter thc supernatanl and Ihc nona dherent cells were decanted and tesled at desired concentrations lor specifit cytolytic reduction
2 Rcsulf> and Interpretation υf Anti Η Υ Killer Cell Activily
Killer cells dircctcd agamsl "seif HLA associated with the minor histocompatibil ity antigen Η Υ can be speufically absorbed by the monolayer F urthcmorc Ihcy can be partitioned into separate populations recognizmg difierenl altced seif HI Α gene products (Goulmy et al l 7)
a Depletionm Cytotoxicity of theHLA-A2RestnctedAntiH-YCytotoxic TCells figure 3 shows thc results of the reduction in lysis after absorption of the HLA A? restncted anti Η Υ cytotoxic effector cells by the appropnate monolayer (ι t the HLA A2 positive male monolayer)
b Reduction m Cytotoxicity of the HLA-A2 and HLA-B7 Restncted Anti Η Υ Cyto toxic Τ Cells
Hgure 4 shows the mdependent absorption of both (A2 Η Υ and B7 Η Υ) subsets of cytotoxic effector cells Complete removal of the HLA A2 restncted anti Η Υ cy toloxicity (see 1 igure 5) was observed when the effector cells (possessing cytotoxic activity directed against the two mdependent phenotypes) recovered from an HLA A2 positive male monolayer were tested against HLA A2 positive male target cells The
113 Unabsorbed absorbed on mon 1 a absorbed on mon 2 absorbed on mon 3 absorbed on mon U
Effector target ratio
I IGURE 4 indcpendtnt dbsorption of the anli HLA Λ2 Η Υ and diiti HLA B7 Η Υ cytotoxiciiy Monolayer 1 HI Α A2 ve B7 -vc d monolayer 2 HL Α A2 + ve B7 ve 9 monolayer 3 HLA Λ2 vc B7 + ve d monolayer 4 - HLA A2 + ve B7 + vc d Tbc unabsorbed and ibsorbcd C TI s arc tested on HLA A2 posi live B7 positive male I irget cclls
100 -i 60 • L0 20
-1
A2B7 A2 B7d* target cells
I IOURL 5 Rcduction in sptcifie cylotoxicity after mono laycr absorption im ib orbed D tnonol lytr Hl Α Α2 i- vc 6 • moiulayer HI Α Β7 4 ve ö *
monolayer HLAA2 ve B7 vc cf
ί «V*
l~-converse effect was seen after absorption of the effector cells on an HLA B7 positive male monolayer thereafter an almost complete depletion of the HLA B7 restneted anti Η Υ cytotoxicity was observed when the effector cells were tested against HLA
B7 positive male target cells (Figure 5)
Partial reduttion in lysis was observed after absorption with either an HLA A2 or an HLA B7 male monolayer and thereafter tested against target cells which carned
114 HLATypmg Methodology and Clwiial Aspctls
I ICiURI 6 RuJuUion in sptufü. tyloioxiuty by addmon of unla btkd 1 tutet eells to Ihe lahcled tar^el teils
fhc lattcr results provide evidence that the anti B7 population can be absorbed in dependently of the anti Λ2 population Effective reduction was also seen after absorp
tion on a monolayer carrying both restncting antigens, A2 and B7 (Figure 4) The removal (by monolayer absorption, Hgures 3 4, and 5) of specific subpopulations ol restnctcd effector cells may bt compatible with the hypotheses of altered seif or dual recognition (7inkernagel el al ) M However, more functional assays are required to rtsolve the question
C Target Cell Inhibition Studies
Unlabeled target cells are able to compete wilh labeled target ceils provided that they express sunilar antigeneic mcmbrane determinants (CML blocking Eechnique50 %3)
Cold target Inhibition studies were performed to confirm the monovalent specificity of the HLA restncted anti Η Υ cytoloxiL Τ ccll> The advantage of the monolayer absorption technique is that subpopulations can be physically removed from cell sus pensions However, (with Special emphasis on effector cells recognuing two "altered" seif H l Α specificities) cold target Inhibition studies result in competitive Inhibition dunng the Lytotoxic 1 cell target cell interaction and theorctically cannot distmguish between polyvalent and monovalent efftetor cells
! Cold färget Inhibition of the HLA A2 Restncted Anti Η Υ Lytotoxit Cells
Figure 6 shows the speufic reduction in cytotoxic activity when 1 x 1(T cold HLA A2 positive male blast cells have been added lo the hol largets Furthermore, Figure 7 shows that the spcufic Hl Α A2 II Υ lysis docs not cross reacl with the serologically cross reacting HI Α Α28 (no Inhibition) Unstimulated lymphocytes (PBLs) are able to inhibit but a 10 fold excess of cells (10") is required to obtain a significant Inhibition
2 C old Idrgct Inhibition ot the Ηί Α A2 dnd HI Α B7 RestnUed Ana Η Υ Cytotoxtt
Cüls
I igurc 8 shows an cxamplc of the icduction in lysis by the addition of selected cold target Inhibitors to the hot target cells No significant Inhibition was obtamed by add
ing 1 χ 10*1 nonrelevant inhibitois, ι e non A2 B7 blasts) whereas 72°7o reduction was
observed by the addnion of cold male target cells carrying both the required HLA A2 and the Ü7 antigen Intcrmcdiatc reduction was observed after the addition of either
115
. 105 hot «2 vc d" brists
^ 105cold Δ?8 ve d*busls
^κ 105cod A2 vn d*PBL
,x TC^codA? vetfbasts
•Sxi04co!d A? ve J1
bei-106cod A2 ve d" FBI JO^cod A? vt tf basl<
J lOURt- 7 Spetific Inhibition by Lold stimulated and unstimulatul largtl uJls lo ihe hot largel cells
)0b U l Α2 Β? d* t! ΙΟ4 ol Α? Β7 d*t
10 t o d Αί Β7 eft Oscod A2 B7 t!1 b
I ICilJRI 8 R^duLdon in lysi by Lold targel cclls
ol the H l Λ Λ2 Η Y/*s7 H Y C T L
D Long-Term Culture and Cloning Procedures
Many developments m cultunng, expanding, and cloning of human Τ cells have been made since it was shown that such cells could bf maintaincd in culture for pro longed penods of timc (Svedmyr) S4 Long term eultare of human Τ iymphocytes can be aclueved by using conditioned mediurn (CM) or Γ cell growth factor (TCGF) both arc denved from the supernatants of mitogen-stünulated Iymphocytes as reported by
scveral authors,55 " and now called lnterleukin 2 (1L 2) The utihzation of irradiated
autologous lymphoblastoid cell line cells improves ihe growth conditions and increases
the cell yield of the eultures (G Bonnard pers jnal communication and Inouye et al 64)
Expansion and maintenance of human alloreactive Τ cell hnes and cloning of either
prohferative or cytotoxic populations also can easily be achieved by using lectins and irradiated feeder cells " 6 9 Using the latler technique we have expanded, maintained, and ctoned the HLA A2 restneted anti Η Υ cytotoxic Τ cells (Goulmy et al 70)
l Long-Term Growth of the HLA-A2 Restnctcd Anti Η Υ Cytotoxic Τ Cell Lines
Continuous growth of 6-day specific cytotoxic effector cells can be carned out by the regulär additions of irradiated feeder cells (ι e , the specific original stimulator cell)
116 HLA Typing Methodology andCiimcal Aspects
Number of effector cells I target ceits χ 10"^ ) I ICURF- 9 Speufn. ant HLA Λ2 Η Υ lysis by Ihree cytotoxic 1 cell ΙιηΐΛ ° ° Δ Δ χ x rcpresem different cytotoxii. Τ cell lincs wilh (heir lyüc capauly agamst HLA A2 positive male
urgct teils I ht Iower lincs in (he figurc (Δ Δ) reprcsenl the dmount
of lysis rcspcUivcly obldiued against HLA A2 negative male targU teils and HI Α Α2 positive ind negative fcmalt. largel teils
and a lectin PHA 1 /jg/mf of the two times crystalli?ed leukoagglutinin Pharmacia 1 ine Chemicdis cat no 17 0630 01 has been used in our studics The eytotoxit Τ teil lines are expanded in lissue eulture flasks (falcon no 3013) containing 2 χ 1(Γ effector
ecüs per ml and arc fed with 3 x 10s 5 000 rad Irradiation feeder cells plus PHA
Lvery ihird or fourth day growih has lo bc checked and the cffeUor cells arc dilulcd
back to a starling number of 2 χ I0s effector cells per mf and are fed Another uscful
teehnique for maintaining the cylotoxic Τ cell iines is ihe use of the commcrually availablc TCOF (Lymphokull Τ Biotest Cat No 812800 an appropnate final con cenlration is 20% in the eulture medium) With Special regard lo the cylotoxic 1 cell Iines (showing HLA A2 restneted anti Η Υ cylolytic aclivity) we have lound thal op timal growih could best bc obtained by utihzing both of the availablc procedurcs In our expenence the maintenance of functional cytotoxic Τ cell lines requires not only TCGF but also repeated antigen prcsentation The cytotoxic Τ cell lines exert stror^, cytolytic activity after 3 weeks in eulture (approximately 50% lysis in a 2 1 eifectoi/ target ratio) and they retain thcir anti male HLA A2 restneted specificiiy (sei l· ifcurt, 9) These ccll lines can bc maintained in eulture for morc than 6 months by ihe usc of mitogen and irradiated feeder cells
2 Clomng
Several clomng procedures can be used Α soft agar technique for separating sub populations from MLC colonies (onginally desenbed by Ro^enszjn et al 7I) is used by 7eevi et al 72 Ceil clomng can bc achieved after 1 x g Sedimentation (as onginally de
scribed by Miller and Philips73) thereafter plated at a dilution of 1 cell/well 6A Clomng
can also be achieved by direct hmitmg dilutions from the long term eultures Limitnig dilutions can be made in such a way that statistically one would expect e g , 10 cells, 1 cell or less than 1 cell per conical microtiter well This latter method has been used in our laboratory with success
Primary hmiung dilutions on a 1 and 10 cell basis per conical microtiter well (250
μί volume) have been set up( and reclomng has been performed as well The hmiting dilutions were prepared from the cytotoxic Τ cell lines (CT lines) Growth promotion is provided by adding specific 3500 rad γ irradiated feeder PBLs (1 x 10* original stim ulator ceüs) and the lectin PHA (1 100 final dilution or l eucoagglutimn 1 mg/ml
117
I IGURL 10 Γ low chart lor long lerm ailtuas tlonint, and rccloi int,
Number of effector cells (target ceils > 10 I IGURF" Π L yd(- cdpaulits ο Γ somc pnmtiy «.olonics dcrviul from
ÜIL firsl linming dilulion agatnsl Hl Λ Α2 positive m"ic tarnet cells
days, the contents of the wells are transferred to separate flat bottomed microtiter
wells (250 μί volume) followcd by the addition of irradiated feeder celh (3 * 104) and
PHA After 3 days, each well IS screencd through the use of a phase contrast micro scope for the presence of growing colomes When growth is observcd the contents of the microwell are transferred to a 2 mi cluster well Cultunng is continued using feeder cells (5 x 10*) and PHA as desenbed above As soön as enough cells can be harvested,
they are tested for cytotoxicity Figure 11 gives some examples of the lytic capacities of some pnmary colomes The results indicatf the vanabihty in the levels of specific cytotoxicity Several hypotheses can be proposed to explain this fan shaped phenome non One possibihty is the existence of hete<rogeneous Τ cell reeeptors in the different
pnmary colonies, each with varying affinuies for the target cells or unequal lytic po tentiais of the pnmary colomes Another explanation for the different lytic potentials could be the result of differences within the pnmary colomes in terms of growth syn chrony Possible objections against the proposed hypothesis is that one cannot refer to "clones" after only one hmiting dilution Therefore, we performed recloning of one pnmary colony (Figure 12, recloning has been done from the pnmary colony in-dicated by an astensk) The results inin-dicated an aimost perfect fit with the earher obtained lytic capacity, which indicates that, now that we have reached the stage in which one might more properly speak of cloning, there is httle Variation in lytic capac ity from one clone to the other
118 HLA Typing Methodology and Chnical Aspects
Pnrnary colomes Cloned cultures
e-~-—"^ Target HLA A2*ve £
Nurnber of effector cells ί target cells χ 10 ώ)
I KiURF 12 Lylic, LapautKA oi somt. retloned tulturcs ong tnally denvcd by Imitting dilution from ihe pnmary colony in dicdted by an astemk and rediulltd on Α one ccll basis
3 tmphtations of Long Term Cultures and Cloning Techmques
7 he possibiltty of maintaining human cytotoxic Τ cells in culture opens up new areas for investigation Homogeneous cloned cultures provide the opportunity for studying different subsets of cytotoxic Τ cells, and their membrane speufic markers and/or receptors biological funtions, and biochemical charactenstics
III R E L E V A N C E I N C L I N I C A L M E D 1 C 1 N E
Τ cell recognition Systems have been described in rodents and have bcen used to clarity Γ effector cell functions (ι c , cell mediated cytotoxic responses towards virus and hapten modified cell surface antigens), 7mkernagel et dl "\ Shearer et al " It is obvious that with the increasing amount of evidence on the functions of the highly polymorphic HLA supergene wc have now come closer to understanding its active role in the immune process, and in all probability, its guidance of the HLA restncted cy totoxic Τ cell subpopulations, e g , in immunosurveillance against tumors and pnmaiy infections However, very httlc evidence has been presented on the presence of HLA restncted cytotoxic Τ cells dunng pnmary viral infections Nevertheless, the involve ment of Η 2 in pnmary viral infections has been established by 7inkernagel and Doh erty74 and Biandcn"
The minor histocompatibilily antigen Η Υ may be involved in graft rejection and acts as a strong transplantation antigen, as was most probably the case in our female bone marrow transplanted aplastic anemia patient This patient rejected an HLA iden tical male graft The presence of strong anti male cytotoxic effector cells and of an anti male HLA restncted IgM antibody (van Lceuwen et ai 7fi) are clearly in vitro re
flections of the already estabhshed influence of sex in both rejec'ion and graft vs host disease in bone marrow transplantation (Storb et al " , van Rood et al 7B)
The importance of the HLA A2 restncted Η V immunity in kidney transplantation was investigated in 1978 (Goulmy et al ) 7 9 Α retrospektive study (Eurotranspiant pa
tients) of cadaveric renal ailograft survivai at two years showed a significant difference belwecn HLA A2 females receivmg HLA A2 male kidneys and non HLA A2 females receiving non HLA A2 male kidneys The Η Υ incompatibihty in donor recipient com binations sharing the HLA A2 antigen resulted in a 38% graft survivai at two years in contrasl lo 58% in the HLA A2 non sharing group
We have recently described another example in which CML, ab an in vitro model, can reflect in vivo processes (Goulmy et al ) 8 0 We have observed a significant correla
119
graft fuiKtion Furthermore compatibihty for the HLA-B locus antigens in the donor recipient pairs and sex-match (i e , a male recipient who received a male graft) predis-pose to the development of donor specific CML nonreactivity
ACKNOWLEDGMENTS
I would like to thank Dr J D'Amaro, Dr C Mawas, Dr Μ J Giphart, and Dr J J van Rood for reading the manuscnpt and for their cntical comments, Ε Blokland for technical expertise, and Α Pesant for typvng the manuscnpt
Work was supported in pari by the Dutch Orgamzation for Health Research (TNO), the Dutch Foundation for Medical Research (FUNGO) which IS subsidized by the Dutch Foundation for the Advancement of Pure Research (7WO), the J Α Cohen Institute for Radiopathoiogy and Radiation Protection (IRS)
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