Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

Université de Montréal

We studied HIV genetic diversity in a cohort of 127 pregnant, HIV-infected women who received prenatal care at Sainte-Justine Hospital in Montreal, Canada, between 1999 and 2003. Clade assignments were derived by phylogenetic analysis of amplified pol sequences. Genotyping was successful in 103 of 127 women, 59 (57.3%) of whom were infected with clade B HIV-1, and 44 (42.7%) with nonclade B viruses, including subtypes A, C, D, F, G, and H. Four sequences remained unassigned. Forty-three of 44 women infected with non-clade B viruses were newcomers from sub-Saharan Africa, and subtype identity was consistent with those circulating in their countries of origin. These results highlight the epidemiologic importance of non-B HIV-1 in antenatal populations in a large North American urban center, underscore the influence of population movements on clade intermixing, and identify a group of patients who could be targeted for surveillance and drug therapy followup.

HIV-1 genetic diversity in antenatal cohort, Canada.

&#x6211;&#x4EEC;&#x7814;&#x7A76;&#x961F;&#x5217;&#x7814;&#x7A76; 1999&#x5E74;&#x81F3; 2003 &#x5E74;&#x5728;&#x52A0;&#x62FF;&#x5927;&#x8499;&#x7279;&#x5229;&#x5C14;&#xFF0C;&#x6536;&#x5230;&#x5723;&#x6770;&#x533B;&#x9662;&#x7684;&#x4EA7;&#x524D;&#x62A4;&#x7406; 127 &#x6000;&#x5B55;&#x3001; &#x611F;&#x67D3;&#x827E;&#x6ECB;&#x75C5;&#x6BD2;&#x7684;&#x5987;&#x5973;&#x4E2D;&#x827E;&#x6ECB;&#x75C5;&#x6BD2;&#x9057;&#x4F20;&#x591A;&#x6837;&#x6027;&#x3002;&#x514B;&#x62C9;&#x5FB7;&#x5206;&#x914D;&#x662F;&#x901A;&#x8FC7;&#x6269;&#x589E;&#x7684; pol &#x5E8F;&#x5217;&#x7684;&#x7CFB;&#x7EDF;&#x53D1;&#x80B2;&#x5206;&#x6790;&#x6D3E;&#x751F;&#x7684;&#x3002;&#x57FA;&#x56E0;&#x5206;&#x578B;&#x662F;&#x6210;&#x529F;&#x7684; 127 &#x540D;&#x5987;&#x5973;&#xFF0C;&#x5176;&#x4E2D; 59 (57.3%) &#x4E0E; B &#x827E;&#x6ECB;&#x75C5;&#x6BD2; 1 &#x514B;&#x62C9;&#x5FB7;&#x53D7;&#x5230;&#x611F;&#x67D3;&#x548C; 44 (42.7%) &#x4E0E; nonclade B &#x75C5;&#x6BD2;&#xFF0C;&#x5176;&#x4E2D;&#x5305;&#x62EC;&#x4E9A;&#x578B; A&#x3001; C&#x3001; D&#x3001; F&#x3001; G &#x548C; H.&#x7B2C; 103 &#x53F7;&#x56DB;&#x4E2A;&#x5E8F;&#x5217;&#x4ECD;&#x672A;&#x5206;&#x914D;&#x3002;&#x56DB;&#x5341;&#x4E09;&#x7684; 44 &#x975E;&#x514B;&#x62C9;&#x5FB7; B &#x75C5;&#x6BD2;&#x611F;&#x67D3;&#x7684;&#x5987;&#x5973;&#x662F;&#x4ECE;&#x6492;&#x54C8;&#x62C9;&#x4EE5;&#x5357;&#x975E;&#x6D32;&#xFF0C;&#x65B0;&#x4EBA;&#x548C;&#x5B50;&#x7C7B;&#x578B;&#x6807;&#x8BC6;&#x662F;&#x4E0E;&#x90A3;&#x4E9B;&#x5728;&#x5176;&#x539F;&#x7C4D;&#x56FD;&#x5FAA;&#x73AF;&#x76F8;&#x4E00;&#x81F4;&#x3002;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x7A81;&#x51FA;&#x663E;&#x793A;&#x7684;&#x975E;-B &#x827E;&#x6ECB;&#x75C5;&#x6BD2;-1 &#x5728;&#x5317;&#x7F8E;&#x5927;&#x57CE;&#x5E02;&#x4E2D;&#x5FC3;&#x7684;&#x4EA7;&#x524D;&#x4EBA;&#x7FA4;&#x4E2D;&#x7684;&#x6D41;&#x884C;&#x75C5;&#x5B66;&#x91CD;&#x8981;&#x6027;&#x3001; &#x5F3A;&#x8C03;&#x5BF9;&#x514B;&#x62C9;&#x5FB7;&#x4E2D;&#x6DF7;&#x7528;&#xFF0C;&#x4EBA;&#x53E3;&#x6D41;&#x52A8;&#x7684;&#x5F71;&#x54CD;&#x548C;&#x6807;&#x8BC6;&#x4E00;&#x7EC4;&#x53EF;&#x4EE5;&#x9488;&#x5BF9;&#x76D1;&#x6D4B;&#x548C;&#x836F;&#x7269;&#x6CBB;&#x7597;&#x968F;&#x8BBF;&#x75C5;&#x4EBA;&#x3002;

&#x827E;&#x6ECB;&#x75C5;&#x6BD2; 1 &#x4EA7;&#x524D;&#x7EC4;&#x7FA4;&#xFF0C;&#x52A0;&#x62FF;&#x5927;&#x7684;&#x9057;&#x4F20;&#x591A;&#x6837;&#x6027;&#x3002;

Nous avons &eacute;tudi&eacute; la diversit&eacute; g&eacute;n&eacute;tique du VIH dans une cohorte de 127 femmes enceintes infect&eacute;es par le VIH qui ont re&ccedil;u des soins pr&eacute;natals &agrave; l&#39;h&ocirc;pital Sainte-Justine &agrave; Montr&eacute;al, au Canada, entre 1999 et 2003. Affectations du clade ont &eacute;t&eacute; obtenues par analyse phylog&eacute;n&eacute;tique des s&eacute;quences amplifi&eacute;es pol. G&eacute;notypage a r&eacute;ussi &agrave; 103 de 127 femmes, 59 (57,3 %) d&#39;entre eux &eacute;taient atteints de clade B du VIH-1 et 44 (42,7 %) avec le virus de le nonclade B, y compris les sous-types A, C, D, F, G et H. Quatre s&eacute;quences est rest&eacute; non attribu&eacute;es. Quarante-trois des 44 femmes infect&eacute;es par le virus B non-clade ont &eacute;t&eacute; les nouveaux arrivants originaires d&#39;Afrique subsaharienne, et sous-type identit&eacute; &eacute;tait conforme &agrave; ceux circulant dans leurs pays d&#39;origine. Ces r&eacute;sultats soulignent l&#39;importance &eacute;pid&eacute;miologique de non B du VIH-1 des populations pr&eacute;natals dans un grand centre urbain nord-am&eacute;ricain, soulignent l&#39;influence des mouvements de population sur le brassage du clade et identifient un groupe de patients qui pourraient &ecirc;tre cibl&eacute;es pour la surveillance et la drogue suivi de th&eacute;rapie.

VIH-1 la diversit&eacute; g&eacute;n&eacute;tique dans la cohorte pr&eacute;natale, Canada.

HIV-1-genetischen Vielfalt in der vorgeburtlichen Kohorte, Kanada.

Abbiamo studiato la diversit&agrave; genetica di HIV in una coorte di 127 donne incinte, HIV-positivi che hanno ricevuto cure prenatali all&#39;ospedale Sainte-Justine a Montreal, in Canada, tra il 1999 e il 2003. Assegnazioni clade erano derivate dall&#39;analisi filogenetica delle sequenze amplificate pol. Genotipizzazione riusciva in 103 127 donne, 59 (57,3%) di cui sono stati infettati con clade B HIV-1, e 44 (42,7%) con virus nonclade B, compresi i sottotipi A, C, D, F, G e H. Quattro sequenze sono rimasti non assegnati. Forty-three di 44 donne infettate con il virus B non clade erano i nuovi arrivati dall&#39;Africa sub-sahariana, e sottotipo identit&agrave; era coerente con quelle circolanti nei loro paesi di origine. Questi risultati sottolineano l&#39;importanza epidemiologica del non - B HIV-1 in popolazioni prenatale in un grande centro urbano nord-americano, sottolineano l&#39;influenza dei movimenti di popolazione su clade mescolanza e identificano un gruppo di pazienti che potrebbero essere mirate per sorveglianza e droga followup di terapia.

Diversit&agrave; genetica di HIV-1 in coorte prenatale, Canada.

&#x6211;&#x3005; &#x306F; 1999 &#x5E74;&#x3068; 2003&#x5E74;&#x306E;&#x9593;&#x306E;&#x30AB;&#x30CA;&#x30C0;&#x306E;&#x30E2;&#x30F3;&#x30C8;&#x30EA;&#x30AA;&#x30FC;&#x30EB;&#x306B;&#x30B5;&#x30F3;&#x30C8;-&#x30B8;&#x30E5;&#x30B9;&#x30C6;&#x30A3;&#x30FC;&#x30CC;&#x75C5;&#x9662;&#x3067;&#x51FA;&#x751F;&#x524D;&#x306E;&#x30B1;&#x30A2;&#x3092;&#x53D7;&#x3051;&#x305F; 127 &#x306E;&#x598A;&#x5A20;&#x3001;HIV &#x611F;&#x67D3;&#x5973;&#x6027;&#x306E;&#x30B3;&#x30DB;&#x30FC;&#x30C8;&#x306B;&#x304A;&#x3051;&#x308B; HIV &#x907A;&#x4F1D;&#x7684;&#x591A;&#x69D8;&#x6027;&#x3092;&#x691C;&#x8A0E;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x30AF;&#x30EC;&#x30FC;&#x30C9;&#x306E;&#x5272;&#x308A;&#x5F53;&#x3066;&#x5897;&#x5E45; pol &#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x306E;&#x7CFB;&#x7D71;&#x89E3;&#x6790;&#x306B;&#x3088;&#x3063;&#x3066;&#x5F97;&#x3089;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x30B8;&#x30A7;&#x30CE;&#x30BF;&#x30A4;&#x30D4;&#x30F3;&#x30B0;&#x306F; 103 &#x306E; 59 (57.3%) &#x4EBA;&#x306E;&#x30AF;&#x30EC;&#x30FC;&#x30C9; B HIV-1 &#x611F;&#x67D3;&#x3057;&#x305F;&#x3001;127 &#x306E;&#x5973;&#x6027;&#x3068; 44 &#xFF08;42.7 &#xFF05;&#xFF09; nonclade B &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x3068;&#x30B5;&#x30D6;&#x30BF;&#x30A4;&#x30D7; A&#x3001;C&#x3001;D&#x3001;F&#x3001;G&#x3001;&#x304A;&#x3088;&#x3073; H &#x3092;&#x542B;&#x3080;&#x3067;&#x6210;&#x529F;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;4 &#x3064;&#x306E;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x306B;&#x5272;&#x308A;&#x5F53;&#x3066;&#x3089;&#x308C;&#x3066;&#x3044;&#x306A;&#x3044;&#x6B8B;&#x3063;&#x305F;&#x3002;40-3 &#x975E;&#x30AF;&#x30EC;&#x30FC;&#x30C9; B &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306B;&#x611F;&#x67D3;&#x3057;&#x305F; 44 &#x5973;&#x6027;&#x306E;&#x30B5;&#x30CF;&#x30E9;&#x4EE5;&#x5357;&#x306E;&#x30A2;&#x30D5;&#x30EA;&#x30AB;&#x304B;&#x3089;&#x306E;&#x65B0;&#x898F;&#x53C2;&#x5165;&#x8005;&#x3068;&#x30B5;&#x30D6;&#x30BF;&#x30A4;&#x30D7;&#x306E;&#x30A2;&#x30A4;&#x30C7;&#x30F3;&#x30C6;&#x30A3;&#x30C6;&#x30A3;&#x306E;&#x8D77;&#x6E90;&#x306E;&#x56FD;&#x3067;&#x5FAA;&#x74B0;&#x3068;&#x4E00;&#x81F4;&#x3060;&#x3063;&#x305F;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D50;&#x679C;&#x306E;&#x975E;-B HIV-1 &#x51FA;&#x751F;&#x524D;&#x96C6;&#x56E3;&#x306F;&#x5927;&#x578B;&#x306E;&#x5317;&#x7C73;&#x306E;&#x90FD;&#x5E02;&#x30BB;&#x30F3;&#x30BF;&#x30FC;&#x3067;&#x75AB;&#x5B66;&#x7684;&#x306A;&#x91CD;&#x8981;&#x6027;&#x3092;&#x5F37;&#x8ABF;&#x3001;&#x4E0A;&#x30AF;&#x30EC;&#x30FC;&#x30C9;&#x6DF7;&#x6676;&#x5316;&#x3001;&#x4EBA;&#x53E3;&#x306E;&#x52D5;&#x304D;&#x306E;&#x5F71;&#x97FF;&#x3092;&#x5F37;&#x8ABF;&#x3059;&#x308B;&#x3001;&#x76E3;&#x8996;&#x3068;&#x85AC;&#x7269;&#x7642;&#x6CD5;&#x306E;&#x30D5;&#x30A9;&#x30ED;&#x30FC; &#x30A2;&#x30C3;&#x30D7;&#x306E;&#x305F;&#x3081;&#x5BFE;&#x8C61;&#x3068;&#x306A;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x60A3;&#x8005;&#x306E;&#x30B0;&#x30EB;&#x30FC;&#x30D7;&#x3092;&#x8B58;&#x5225;&#x3057;&#x307E;&#x3059;&#x3002;

HIV 1 &#x907A;&#x4F1D;&#x7684;&#x591A;&#x69D8;&#x6027;&#x51FA;&#x751F;&#x30B3;&#x30DB;&#x30FC;&#x30C8;&#x3001;&#x30AB;&#x30CA;&#x30C0;&#x3002;

&#xC6B0;&#xB9AC;&#xAC00; 1999&#xC5D0;&#xC11C; 2003 &#xC0AC;&#xC774; &#xCE90;&#xB098;&#xB2E4; &#xBAAC;&#xD2B8;&#xB9AC;&#xC62C; &#xC14D; &#xD2B8;-&#xC950;&#xC2A4;&#xD2F4; &#xBCD1;&#xC6D0;&#xC5D0;&#xC11C; &#xCD9C;&#xC0DD; &#xC804; &#xCF00;&#xC5B4;&#xB97C; &#xBC1B;&#xC740; 127 &#xC784;&#xC2E0;, &#xC5D0;&#xC774;&#xC988;&#xC5D0; &#xAC10;&#xC5FC; &#xB41C; &#xC5EC;&#xC790;&#xC758; &#xCF54; &#xD638;&#xD2B8; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C; HIV &#xC720;&#xC804;&#xC790; &#xB2E4;&#xC591;&#xC131;&#xC744; &#xACF5;&#xBD80; &#xD588;&#xB2E4;. Clade &#xD560;&#xB2F9;&#xC758; &#xC99D;&#xD3ED;&#xB41C; pol &#xC2DC;&#xD000;&#xC2A4; phylogenetic &#xBD84;&#xC11D;&#xC5D0; &#xC758;&#xD574; &#xD30C;&#xC0DD; &#xB418;&#xC5C8;&#xB2E4;. &#xC720;&#xC804;&#xD615; 127 &#xC5EC;&#xC131;, 59 (57.3%) &#xBA85;&#xC740; clade B &#xC5D0;&#xC774;&#xC988;-1 &#xAC10;&#xC5FC; &#xB418;&#xC5C8;&#xB2E4;, &#xADF8;&#xB9AC;&#xACE0; nonclade B &#xBC14;&#xC774;&#xB7EC;&#xC2A4;, A, C, D, F, G &#xBC0F; H. &#xD558;&#xC704;&#xB97C; &#xD3EC;&#xD568; &#xD55C; 44 (42.7%)&#xC758; 103&#xC5D0; &#xC131;&#xACF5; &#xD588;&#xB2E4; 4 &#xC2DC;&#xD000;&#xC2A4;&#xC5D0; &#xD560;&#xB2F9; &#xB418;&#xC9C0; &#xC54A;&#xC740; &#xB0A8;&#xC544; &#xC788;&#xC5C8;&#xB2E4;. 44 &#xC5EC;&#xC131;&#xC758; &#xBE44; clade B &#xBC14;&#xC774;&#xB7EC;&#xC2A4;&#xC5D0; &#xAC10;&#xC5FC; &#xB41C; 40-3 &#xC0AC;&#xD558;&#xB77C; &#xC0AC;&#xB9C9; &#xC774;&#xB0A8;&#xC758; &#xC544;&#xD504;&#xB9AC;&#xCE74;&#xC5D0;&#xC11C; &#xC774;&#xBBFC;&#xC790; &#xB418;&#xC5C8;&#xACE0; &#xD558;&#xC704; &#xC2E0;&#xBD84; &#xAE30;&#xC6D0;&#xC758; &#xADF8;&#xB4E4;&#xC758; &#xB098;&#xB77C;&#xC5D0;&#xC11C; &#xC21C;&#xD658;&#xC640; &#xC77C;&#xCE58; &#xD588;&#xB2E4;. &#xC774; &#xACB0;&#xACFC; &#xC5ED;&#xD559;&#xC801; &#xC911;&#xC694;&#xC131;&#xC758; &#xBE44;-B V-1 &#xD070; &#xBD81;&#xBBF8; &#xB3C4;&#xC2DC; &#xC13C;&#xD130;&#xC5D0;&#xC11C; &#xC0B0; &#xC778;&#xAD6C;&#xC5D0; &#xAC15;&#xC870; &#xD45C;&#xC2DC; &#xD558; &#xACE0; &#xBC11;&#xC904; clade &#xD63C;&#xD569;&#xACFC;&#xC5D0; &#xC778;&#xAD6C; &#xC6B4;&#xB3D9;&#xC758; &#xC601;&#xD5A5; &#xAC10;&#xC2DC; &#xBC0F; &#xC57D;&#xBB3C; &#xCE58;&#xB8CC; &#xCD94;&#xAC00; &#xB2F5;&#xBCC0;&#xC5D0; &#xB300; &#xD55C; &#xD0C0;&#xAC9F;&#xC774; &#xB420; &#xC218; &#xC788;&#xB294; &#xD658;&#xC790;&#xC758; &#xADF8;&#xB8F9;&#xC744; &#xC2DD;&#xBCC4;.

&#xC5D0;&#xC774;&#xC988;-1 &#xC0B0; &#xC77C;&#xB300;, &#xCE90;&#xB098;&#xB2E4;&#xC5D0;&#xC5D0;&#xC11C; &#xC720;&#xC804;&#xC801; &#xB2E4;&#xC591;&#xC131;.

Estudiamos la diversidad gen&eacute;tica del VIH en una cohorte de 127 mujeres embarazadas, infectados por el VIH que recibieron atenci&oacute;n prenatal en el Hospital Sainte-Justine en Montreal, Canad&aacute;, entre 1999 y 2003. Asignaciones de clade se derivaron por an&aacute;lisis filogen&eacute;tico de secuencias amplificadas pol. Genotipado logr&oacute; 103 de 127 mujeres, 59 (57.3%) de los cuales estaban infectados con clade B VIH-1 y 44 (42,7%) con el virus de la nonclade B, incluyendo los subtipos A, C, D, F, G y H. Cuatro secuencias permanec&iacute;an sin asignar. Cuarenta y tres de 44 mujeres infectadas con el virus de no-clado B fueron reci&eacute;n llegados de &Aacute;frica subsahariana, y subtipo identidad era constante con aquellos que circulan en sus pa&iacute;ses de origen. Estos resultados destacan la importancia epidemiol&oacute;gica del no - B VIH-1 en poblaciones prenatales en un gran centro urbano norteamericano, subrayan la influencia de los movimientos de poblaci&oacute;n en clade amasar e identifican un grupo de pacientes que podr&iacute;an ser destinados a la vigilancia y drogas seguimiento de la terapia.

Diversidad gen&eacute;tica del VIH-1 en la cohorte prenatal, Canad&aacute;.

Therapies based on enhancing the numbers and/or function of T regulatory cells (Tregs) represent one of the most promising approaches to restoring tolerance in many immune-mediated diseases. Several groups have investigated whether human Tregs suitable for cellular therapy can be obtained by in vitro expansion, in vitro conversion of conventional T cells into Tregs, or gene transfer of the FOXP3 transcription factor. To date, however, none of these approaches has resulted in a homogeneous and stable population of cells that is as potently suppressive as ex vivo Tregs. We developed a lentivirus-based strategy to ectopically express high levels of FOXP3 that do not fluctuate with the state of T-cell activation. This method consistently results in the development of suppressive cells that are as potent as Tregs and can be propagated as a homogeneous population. Moreover, using this system, both naïve and memory CD4(+) T cells can be efficiently converted into Tregs. To date, this is the most efficient and reliable protocol for generating large numbers of suppressive CD4(+) Tregs, which can be used for further biological study and developed for antigen-specific cellular therapy applications.

Generation of potent and stable human CD4+ T regulatory cells by activation-independent expression of FOXP3.

Umbilical cord blood (UCB) is increasingly used as a source of hematopoietic progenitor cells to treat a variety of disorders. UCB transplant is associated with comparatively reduced incidence of graft-versus-host disease, robust graft versus leukemia effect, and relatively high incidence of opportunistic infections, three processes in which donor-derived T lymphocytes are known to be predominantly involved. To examine the differential functionality of UCB T cells, CD8(+) T cells specific for the melanoma-associated HLA-A2-restricted Melan-A(26-35) A27L peptide were isolated from HLA-A2(+) and HLA-A2(-) UCB samples and HLA-A2(+) and HLA-A2(-) adult peripheral blood using A2/Melan-A tetramers. In UCB samples, A2/Melan-A(+) CD8(+) T cells were detected at a frequency of 0.04%, were more frequent in HLA-A2(+) UCB, and were polyclonal and mostly naive. Consistent with Ag-driven expansion, the frequency of A2/Melan-A(+) CD8(+) T cells was increased following stimulation with cognate peptide or polyclonal activation, they acquired cell-surface markers reflective of effector/memory differentiation, their TCR repertoire became oligoclonal, and they expressed cytolytic activity and produced IFN-gamma. Although functional properties of A2/Melan-A(+) CD8(+) T cells derived from HLA-A2(+) UCB resembled those of HLA-A2(+) adult peripheral blood, they were more likely to reach terminal differentiation following polyclonal stimulation and produced less IFN-gamma in response to cognate peptide. A2/Melan-A(+) CD8(+) T cells from HLA-A2(-) UCB were poorly cytolytic, produced little IFN-gamma, and were predominantly monofunctional or nonfunctional. These properties of UCB-derived CD8(+) T cells could contribute to the reduced incidence of graft-versus-host disease and heightened incidence of opportunistic infections observed following UCB transplant.

Umbilical cord blood T cells respond against the Melan-A/MART-1 tumor antigen and exhibit reduced alloreactivity as compared with adult blood-derived T cells.

Near full-length genomes of 4 unclassified HIV-1 variants infecting patients enrolled in an antenatal cohort in Canada were obtained by sequencing. All 4 variants showed original recombination profiles, including A1/A2/J, A1/D, and A1/G/J/CRF11_cpx structures. Identification of these variants highlights the growing prevalence of unique recombinant forms of HIV-1 in North America.

Novel HIV-1 recombinant forms in antenatal cohort, Montreal, Quebec, Canada.

UCBT has been used for almost 25 years to treat a variety of malignant and nonmalignant childhood diseases. The biological properties of NK cells and T cells and their implication in engraftment, immune reconstitution, OIs, leukemic relapse, and GvHD have been explored in the context of UCBT. These studies have established that lymphocytes have a major impact on the outcome of UCBT and that NK cells and T cells play complementary and contrasting roles in immune reconstitution and the GvL effect. Therefore, novel strategies to improve the outcome of UCBT recipients, including immunotherapeutic regimens, should be based on key immunologic features of UCB T lymphocytes and NK cells.

Complementary and contrasting roles of NK cells and T cells in pediatric umbilical cord blood transplantation.

Recipients of umbilical cord blood (UCB) transplantation (UCBT) face a high risk of morbidity and mortality related to opportunistic infections (OI) and leukemic relapse. To understand the molecular basis of these UCBT-related complications, the characteristics of UCB-derived antigen-specific CD8(+) T cells were examined in a group of pediatric UCBT recipients. Compared with the UCB graft inoculum and the late post-UCBT period (12-36 months), declining clonal diversity of UCB-derived CD8(+) T cells specific for the Melan-A(26-35) A27L peptide and high frequencies of PD-1-expressing CD8(+) T cells were observed in the first 3 months after UCBT, a period during which OIs are most frequent. The CD8(+) T-cell compartment predominantly comprised CD45RA(+) CCR7(-) terminally differentiated effector-memory T cells until 6 months after UCBT, at which time the polyfunctionality of antigen-specific CD8(+) T cells was reestablished. Finally, the frequency of PD-1(+) CD8(+) T cells was significantly higher in subjects who subsequently experienced leukemic relapse. This study informs the biologic properties of UCB-derived CD8(+) T cells and provides a rationale for the characteristics of UCBT in terms of immune reconstitution and OI. These results also suggest that the elevated frequency of PD-1(+) CD8(+) T cells could be associated with leukemic relapse in pediatric UCBT recipients.

CD8(+) T-cell reconstitution in recipients of umbilical cord blood transplantation and characteristics associated with leukemic relapse.

Plasmacytoid dendritic cells (PDCs) from human umbilical cord blood (UCB) produce lower amounts of IFN-α upon TLR stimulation compared with adult counterparts. This difference may play a role in the low graft-versus-host disease rate after UCB transplantation and in the impaired immune response of the neonate to pathogens. Comparing UCB PDC to their adults counterparts, we found that they exhibited a mature surface phenotype and a normal antigen uptake. They upregulated costimulatory molecules upon activation, although with delayed kinetics. Protein, but not ARN, levels of TLR-9, MyD88, IRAK1 and IRF-7, involved in the TLR-9 signaling pathway were reduced. The expression levels of miR-146a and miR-155, known to be involved in the post-transcriptional down-regulation of immune responses, were higher. These data point out a post-transcriptional down-regulation of the TLR-9/IRF-7 signaling pathway in UCB PDC.

Post-transcriptional down-regulation of Toll-like receptor signaling pathway in umbilical cord blood plasmacytoid dendritic cells.

CMV and varicella zoster virus (VZV) are significant causes of morbidity and mortality following umbilical cord blood transplantation (UCBT). However, the kinetics of reconstitution and protective potential of antiviral cell-mediated immune responses following UCBT remain poorly characterized. In this study, the reconstitution of CMV- and VZV-specific T cell responses was assessed using IFN-γ ELISPOT in 28 children who underwent UCBT to treat hematological or inherited disorders. Barely detectable in the first 3 mo posttransplantation, CMV- and VZV-specific T cell responses were observed in 30.4% and 40.3% of study subjects after 36 mo of follow-up. Four of five CMV-seropositive subjects developed detectable levels of circulating CMV DNA (DNAemia), and 5 of 17 VZV-seropositive patients experienced herpes zoster during the posttransplant period. Four CMV-seronegative subjects developed IFN-γ responses against CMV, and four subjects developed a VZV-specific IFN-γ response without clinical signs of infection. No CMV- or VZV-related events were observed in study subjects following the development of CMV- or VZV-specific responses > 150 spot-forming units/10(6) PBMCs, consistent with T cell-mediated protection. Finally, famciclovir prophylaxis did not strictly prevent the reconstitution of the VZV-specific T cell repertoire, because the frequency of T cells producing IFN-γ in response to VZV Ags reached levels consistent with protection in two nonzoster subjects. Monitoring of CMV- and VZV-specific cell-mediated immunity could inform immunocompetence and guide the initiation and cessation of antiherpetic prophylaxis in UCBT recipients.

Reconstitution of protective immune responses against cytomegalovirus and varicella zoster virus does not require disease development in pediatric recipients of umbilical cord blood transplantation.

Coreceptor switch from CCR5 to CXCR4 is associated with HIV disease progression. To document the evolution of coreceptor tropism during pregnancy, a longitudinal study of envelope gene sequences was performed in a group of pregnant women infected with HIV-1 of clade B (n=10) or non-B (n=9). Polymerase chain reaction (PCR) amplification of the V1-V3 region was performed on plasma viral RNA, followed by cloning and sequencing. Using geno2pheno and PSSMX4R5, the presence of X4 variants was predicted in nine of 19 subjects (X4 subjects) independent of HIV-1 clade. Six of nine X4 subjects exhibited CD4(+) T cell counts <200 cells/mm(3), and the presence of X4-capable virus was confirmed using a recombinant phenotypic assay in four of seven cases where testing was successful. In five of nine X4 subjects, a statistically significant decline in the geno2pheno false-positive rate was observed during the course of pregnancy, invariably accompanied by progressive increases in the PSSMX4R5 score, the net charge of V3, and the relative representation of X4 sequences. Evolution toward X4 tropism was also echoed in the primary structure of V2, as an accumulation of substitutions associated with CXCR4 tropism was seen in X4 subjects. Results from these experiments provide the first evidence of the ongoing evolution of coreceptor utilization from CCR5 to CXCR4 during pregnancy in a significant fraction of HIV-infected women. These results inform changes in host-pathogen interactions that lead to a directional shaping of viral populations and viral tropism during pregnancy, and provide insights into the biology of HIV transmission from mother to child.

Natacha Merindol

Université de Montréal

Development of an IFN-γ ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation