HLAMatchmaker-Based Analysis of MICA Antibody Reactivity PatternsHLAMatchmaker-Based Analysis of MICA Antibody Reactivity PatternsRene J. Duquesnoy1, Justin W. Mostecki2, Jayasree Hariharan2, Kristi Colacioppo2, Bryan Ray2 and Ivan Balazs2

1University of Pittsburgh Medical Center and 2Tepnel Lifecodes CorporationIntroductionHLAMatchmaker is a computer algorithm that determines HLA compatibility at the structural level. Donor-recipient histocompatibility is assessed with polymorphic amino acid configurations that represent structurally defined elements of HLA epitopes. The term “eplet” represents an epitope defined by a polymorphic surface residue and other residues within a radius of 3.0-3.5 Ångstroms. Structurally based HLA matching reduces humoral allosensitization and correlates with good transplant outcome. Moreover, HLAMatchmaker is useful in the antibody analysis of sera from highly sensitized patients.Recent studies have suggested a clinical significance of anti-MICA antibodies in transplantation. We have developed an eplet-based version HLAMatchmaker to assess the epitope specificity of anti-MICA antibodies. The amino acid sequences of the MICA alleles contain multiple polymorphic positions that likely contribute to the epitope repertoire of MICA. Molecular viewing of two published 3D structures of MICA (PDB codes IB35 and 1HYR downloaded from the NCBI website (http://www.ncbi.nlm.nih.gov/Structure) has identified 30 polymorphic positions on 53 MICA alleles. Figure1 shows four views of the polymorphic residues on MICA. Table 1 shows the locations of polymorphic residues and the residue positions used to determine MICA eplets. Table 2 shows MICA alleles with the more common eplets.

HLAMatchmaker Analysis of MICA Antibodies: Four Examples

1. This patient typed as A*008, *010 and had received a kidney transplant from a sister who was mismatched for A*012 which had 10 mismatched eplets. Although the serum showed weak anti-MICA reactivity with this small panel it became apparent that the antibodies are specific for 24TC. 24TC containing antigens are shown in black.

2. This highly reactive serum was from a patient who typed as A*012. In this 20 antigen panel only one non-self antigen A*043 was negative. All 18 reactive antigens shared the 156H eplet and are shaded in black.

3. No MICA typing information was available. This serum reacted only with 14G positive antigens shown in black. The “acceptable” and “unacceptable “ alleles are listed.

4. No MICA typing information was available. Although this serum reacted with all 14W positive antigens (shown by black and hatched bars) you will note that there was strong reactivity with the antigens that shared 24AY, 129V, 173EE, and 206S2 which are shaded in black. It seems likely that the antibody specificity is against 14W plus one or more of these eplets. Absorption/elution analysis with informative alleles may provide further insight.

ConclusionsThe HLAMatchmaker program to analyze MICA antibodies can be downloaded from the website http://www.HLAMatchmaker.net which has also other programs and articles about structurally based histocompatibility. Please contact Duquesnoyr@upmc.edu if you have any questions or comments. This study was supported by NIH grant AI-55933

Figure 1 Four views of polymorphic residues on MICA

Table 1 Location and Surface Expression ofPolymorphic Positions on MICA Molecules Table 2 More Common Eplets on MICA Alleles

1stMICA 2ndMICAPatient A*012 xIMM x xPotential Donorx x

Alleles that give negative reactions:A*043

Reactive eplet:156H non*012*021*032*043

CutOffPrep# MICA SELF <400 MFI #Ep Remaining mm EpletsCON1 Neg cont 146 ,,,,,Probe 301 A*001 1298 2 ,,,,,,,,,,,125K2,,,,,156H,,,,,,,,,,,,,,Probe 302 A*002 2602 2 ,14G,,,,,,,,,,,,,,,156H,,,,,,,,,,,,,,Probe 304 A*004 1835 10 ,,24AY,,,,,,,122VQ,,,,129V,,,156H,173EE,175SE,174EES,181R,206S2,,,214TT,,,,,,Probe 306 A*006 1171 9 ,,24AY,,,,,,,122VQ,,,,129V,,,156H,173EE,175SE,174EES,,206S2,,,214TT,,,,,,Probe 307 A*007 6008 1 ,,,,,,,,,,,,,,,,156H,,,,,,,,,,,,,,Probe 308 A*008 1837 8 ,,24AY,,,,,,,,,,,129V,,,156H,173EE,,174EEG,,206S2,,213I,,,251R,,,,Probe 309 A*009 1357 9 ,,24AY,,,,,,,122VQ,,,,129V,,,156H,173EE,175SE,174EES,,206S2,,,214TT,,,,,,Probe 311 A*011 1700 4 ,14G,,,,,,,,,,,,,,151V,156H,,,,,,,,,,,,,271A,Probe 312 A*012 SELF 238 - ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 315 A*015 1877 3 ,14G,,,,,,,114R,,,,,,,,156H,,,,,,,,,,,,,,Probe 316 A*016 1050 10 ,,24AY,,,,,,,,,,,129V,,,156H,173EE,175SE,174EES,,206S2,,213I,,221L,251R,,,,Probe 317 A*017 1629 3 ,14G,,,,,,,,122LR,,,,,,,156H,,,,,,,,,,,,,,Probe 318 A*018 5565 1 ,,,,,,,,,,,,,,,,156H,,,,,,,,,,,,,,Probe 319 A*019 1311 9 ,,24AY,,,,,,,,,,,129V,,,156H,173EE,175SE,174EES,,206S2,,213I,,,251R,,,,Probe 329 A*029 4404 2 ,,,,,,,,,,,,,,142I,,156H,,,,,,,,,,,,,,Probe 330 A*030 2317 3 ,14G,,,,,,,,,,,,,,,156H,,,,,,,,,,,,,271A,Probe 333 A*033 1319 10 ,,24AY,,,,,,,,124S,,,129V,,,156H,173EE,175SE,174EES,,206S2,,213I,,,251R,,,,Probe 343 A*043 NEG 256 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 346 A*046 1709 3 ,14G,,,,,,,,,,,,,,,156H,,,,,,208GCW,,,,,,,,Probe 350 A*050 2012 3 ,14G,,,,,90FQ,,,,,,,,,,156H,,,,,,,,,,,,,,Probe 77 Pos cont 12249 ,,,,,

1stMICA 2ndMICAPatient Unknown UnknownIMM Unknown Unknown

Alleles that give negative reactions:A*002 A*017A*011 A*046A*015 A*050

Reactive eplets Unacceptable Alleles14W *001*004*005*006*007*008*009*010*012*016*018*019*021*024*025*026*027*028*029*031*032*033

*037*038*039*040*042*043*045*048*049*05124AY *004*005*006*008*009*010*016*019*024*025*027*028*031*032*033*042*044*048*049*051129V *004*005*006*008*009*010*013*016*019*022*024*027*028*033*044*048*049*053173EE *004*006*008*009*010*013*014*016*019*022*024*027*028*033*036*044*048*049*053206S2 *004*005*006*008*009*010*016*019*022*024*027*033*034*035*037*038*039*042*044*048*049*053

Alleles in bold font are not found in the 14W group

Cut-offPrep # MICA <800 MFI #Ep Remaining mm EpletsCON1 x NEG 192 ,,,,,Probe 301 A*001 2413 3 ,14W,24TC,,,,,,,,,125K2,,,,,,,,,,,,,,,,,,,Probe 302 A*002 NEG 657 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 304 A*004 17507* 10 ,14W,24AY,,,,,,,122VQ,,,,129V,,,,173EE,175SE,174EES,181R,206S2,,,214TT,,,,,,Probe 306 A*006 15961* 9 ,14W,24AY,,,,,,,122VQ,,,,129V,,,,173EE,175SE,174EES,,206S2,,,214TT,,,,,,Probe 307 A*007 5036 1 ,14W,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 308 A*008 17983* 8 ,14W,24AY,,,,,,,,,,,129V,,,,173EE,,174EEG,,206S2,,213I,,,251R,,,,Probe 309 A*009 16477* 9 ,14W,24AY,,,,,,,122VQ,,,,129V,,,,173EE,175SE,174EES,,206S2,,,214TT,,,,,,Probe 311 A*011 NEG 693 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 312 A*012 2777 3 ,14W,24TC,,,,,,,,,,,,,,156L,,,,,,,,,,,,,,Probe 315 A*015 NEG 602 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 316 A*016 16615* 10 ,14W,24AY,,,,,,,,,,,129V,,,,173EE,175SE,174EES,,206S2,,213I,,221L,251R,,,,Probe 317 A*017 NEG 545 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 318 A*018 3062 2 ,14W,24TC,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 319 A*019 16545* 9 ,14W,24AY,,,,,,,,,,,129V,,,,173EE,175SE,174EES,,206S2,,213I,,,251R,,,,Probe 329 A*029 5034 2 ,14W,,,,,,,,,,,,,142I,,,,,,,,,,,,,,,,Probe 330 A*030 NEG 648 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 333 A*033 16922* 10 ,14W,24AY,,,,,,,,124S,,,129V,,,,173EE,175SE,174EES,,206S2,,213I,,,251R,,,,Probe 343 A*043 5039 2 ,14W,,,,,,,,,,,,,,,156R,,,,,,,,,,,,,,Probe 346 A*046 NEG 760 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 350 A*050 NEG 588 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 77 PosCont 17006 ,,,,,

*Strong reactions if allele has also 24AY,129V, 173EE and 206S2Which eplet(s) react with antibody?

1stMICA 2ndMICAPatient A*008 A*010IMM A*010 A*012

Alleles that give negative reactions:A*004A*009 IMM Remaining Donor Eplets Total Mm EpletsA*019 A*010 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,A*002 A*012 ,,24TC,,,,,,,,,,,,,,156L,,,,,,,,,,,,,, ,,24TC,,,36C,,,,,,,,129M,,,156L,173KE,,174KEG,,206G2,208GYW,213T,,,251Q,,,,

ReactiveEplet Unacceptable Alleles24TC *001*012*018*021

CutOffPrep # AmountMICA SELF IMM <450 MFI #Ep Remaining mm Eplets Total Mm EpletsCON1 Neg Cont 238 ,,,,, ,,,,,Probe 301 0.90 A*001 1021 2 ,,24TC,,,,,,,,,125K2,,,,,,,,,,,,,,,,,,,,,24TC,,,36C,,,,,,125K2,0,129M,,,,0,0,0,,206G2,208GYW,213T,,,251Q,,,,Probe 302 0.92 A*002 NEG 440 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, ,14G,24AC,,25AVC,36C,,,,,,,,129M,,,,173KE,,174KEG,,206G2,208GYW,213T,,,251Q,,,,Probe 304 0.95 A*004 NEG 290 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, ,,,,,,,,,122VQ,,,0,,,,,,,,181R,,,213T,214TT,,251Q,,,,Probe 307 0.93 A*007 NEG 403 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, ,,24AC,,25AVC,36C,,,,,,,,129M,,,,173KE,,174KEG,,206G2,208GYW,213T,,,251Q,,,,Probe 308 0.85 A*008 SELF NEG 209 - ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 309 1.00 A*009 NEG 283 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, ,,,,,,,,,122VQ,,,0,,,,,,,,,,,213T,214TT,,251Q,,,,Probe 310 0.24 A*010 SELF IMM NEG 229 - ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 312 0.94 A*012 IMM 996 2 ,,24TC,,,,,,,,,,,,,,156L,,,,,,,,,,,,,, ,,24TC,,,36C,,,,,,,,129M,,,156L,173KE,,174KEG,,206G2,208GYW,213T,,,251Q,,,,Probe 318 0.94 A*018 1089 1 ,,24TC,,,,,,,,,,,,,,,,,,,,,,,,,,,, ,,24TC,,,36C,,,,,,,,129M,,,,173KE,,174KEG,,206G2,208GYW,213T,,,251Q,,,,Probe 319 1.00 A*019 NEG 268 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 77 Pos Cont 18528

Position Location Surface Exposure Eplet Positions6 underside + 614 side ++ 1424 side + 24 3624 side + 24 26 3626 side + 2636 side + 3691 underside + 90 91

105 side ++ 105114 under ++ 114122 side + 122 91122 side + 122 124 125124 side ++ 124125 side ++ 125129 side ++ 129142 top + 142151 top ++ 151156 top ++ 156173 side ++ 173 125173 side ++ 173 125 175175 side + 175 125181 side + 181206 side + 206208 side + 206 208 210210 side ++ 210213 side ++ 213 215215 side + 215221 side ++ 221251 bottom ++ 251256 bottom ++ 256268 side + 268271 side ++ 271

EPLET MICA Alleles

6P *010*0256R non*010*02514G *002*011*013*014*015*017*020*022*023*030*034*035*036*041*044*046*047*050*052*05314W *001*004*005*006*007*008*009*010*012*016*018*019*021*024*025*026*027*028*029*031*032*033*037*038*039*040*042*043*045*048*049*05124AC *002*007*011*013*014*015*017*020*022*023*026*029*030*034*035*036*037*038*039*040*041*043*045*046*047*050*052*05324AY *004*005*006*008*009*010*016*019*024*025*027*028*031*032*033*042*044*048*049*05124TC *001*012*018*02125AVC *002*007*011*013*014*015*017*020*022*023*026*029*030*034*035*036*037*038*039*040*043*045*046*050*052*05336C *001*002*007*011*012*013*014*015*017*018*020*021*022*023*026*029*030*034*035*036*037*038*039*040*041*043*045*046*047*050*052*053114G non*014*015114R *014*015122LQ non*004*006*009*017*044*049122LR *017122VQ *004*006*009*044*049123LTE non*004*006*009*044*049125E non*001*031*040125K2 *001*031*040129M *001*002*007*011*012*014*015*017*018*020*021*023*025*026*029*030*031*032*034*035*036*037*038*039*040*041*042*043*045*046*047*050*051*052129V *004*005*006*008*009*010*013*016*019*022*024*027*028*033*044*048*049*053151M non*011*034151V *011*034156H non*012*021*032*043156L *012*021*032173EE *004*006*008*009*010*013*014*016*019*022*024*027*028*033*036*044*048*049*053173KE *002*005*007*011*012*015*017*018*020*021*023*025*026*029*030*032*034*035*037*038*039*041*042*043*045*046*047*050*051*052174EEG *008*013*014*024*027*028*048*053174EES *004*006*009*010*016*019*022*033*036*044*049174KEG *002*005*007*011*012*015*017*018*020*021*023*025*026*029*030*034*035*037*038*039*041*042*043*045*046*047*050*051*052174KKS *001175GE *002*005*007*008*011*012*013*014*015*017*018*020*021*023*024*025*026*027*028*029*030*034*035*037*038*039*041*042*043*045*046*047*048*050*051*052*053175SE *004*006*009*010*016*019*022*032*033*036*044*049181R *004*014*032*044181T non*004*014*032*044206G2 *001*002*007*011*012*013*014*015*017*018*020*021*023*025*026*028*029*030*031*032*036*040*041*043*045*046*047*050*051*052206S2 *004*005*006*008*009*010*016*019*022*024*027*033*034*035*037*038*039*042*044*048*049*053208GYW *001*002*007*011*012*013*014*015*017*018*020*021*023*025*026*028*029*030*031*032*036*040*041*043*045*047*050*051*052213I *008*010*016*019*022*027*033*035*037*039*042*048*053213T *001*002*004*005*006*007*009*011*012*013*014*015*017*018*020*021*023*024*025*026*028*029*030*031*032*034*036*038*040*041*043*044*045*046*047*049*050*051*052214TT *004*005*006*009*024*034*038*044*049221L *016*039221V non*016*039251Q *001*002*004*006*007*009*011*012*014*015*017*018*020*021*023*024*025*026*028*029*030*031*032*034*036*038*040*041*043*044*046*047*049*050*051*052251R *005*008*010*013*016*019*022*027*033*035*037*039*042*048*053271A *011*030*047271P non*011*030*047

Example 1

Example 2

Example 4

1stMICA 2ndMICAPatient Unknown UnknownIMM Unknown Unknown

MICA Alleles that give negative reactions:

A*001 A*010A*004 A*012A*006 A*016A*007 A*029A*008 A*033A*009 A*043

Reactive eplet Unacceptable MICA alleles14G *002*011*013*014*015*017*020*022*023*030*034*035*036*041 *002*011*013*014*015*017*020*022*023*030*034*035*036*041*044*046*047*050*052*053

*044*046*047*050*052*053Acceptable Alleles: *001*004*005*006*007*008*009*010*012*016*018*019*021*024*025*026*027*028*029*031*033*037*038*039*040*042*043*048*049*051*032*045*052

CutOffPrep# MICA <300 MFI #Ep Eplets on Reactive AllelesCON1 Neg Cont NEG 158 ,,,,,Probe 301 A*001 NEG 195 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 302 A*002 1511 1 ,14G,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 304 A*004 NEG 236 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 306 A*006 NEG 281 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 307 A*007 NEG 229 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 308 A*008 NEG 233 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 309 A*009 NEG 224 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 311 A*011 1217 3 ,14G,,,,,,,,,,,,,,151V,,,,,,,,,,,,,,271A,Probe 312 A*012 NEG 166 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 315 A*015 1442 2 ,14G,,,,,,,114R,,,,,,,,,,,,,,,,,,,,,,Probe 316 A*016 NEG 168 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 317 A*017 1290 2 ,14G,,,,,,,,122LR,,,,,,,,,,,,,,,,,,,,,Probe 318 A*018 NEG 165 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 319 A*019 NEG 178 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 329 A*029 NEG 229 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 330 A*030 1544 2 ,14G,,,,,,,,,,,,,,,,,,,,,,,,,,,,271A,Probe 333 A*033 NEG 219 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 343 A*043 NEG 241 0 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Probe 346 A*046 1948 2 ,14G,,,,,,,,,,,,,,,,,,,,,208GCW,,,,,,,,Probe 350 A*050 1398 2 ,14G,,,,,90FQ,,,,,,,,,,,,,,,,,,,,,,,,Probe 77 PosCont 20649 ,,,,,

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