HLA-A, -B restriction of cytotoxic T cells.


Full text


HL Α Typing:



Clinical Aspects

Volume II


Soldano Ferrone, M.D., Ph.D.


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


CRC Press

Boca Raton,




Chapter 10


E. Goulmy


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 1

Cr 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


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



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


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)



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


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/


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


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





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


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



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 •




A2B7 A2 B7

d* 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




. 105 hot «2 vc d" brists ^ 105cold Δ?8 ve d*busls

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)


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



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


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



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


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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