Fry, T.J.

Mackall, C.L.

Melchionda, F.

English

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granules containing perforin. Products treated with LLME dem-onstrated a median depletion of 92% of CD56 cells, 67 % ofCD8 cells, but only 37% of CD4 cells. Patients initially re-ceived 10e4 (haploidentical donor, n  3), 10e5 (HLA identicalunrelated donor, n  1), 10e6 (HLA identical sibling donor, n 4), or 10e7 (HLA identical sibling donor, n  1) CD3 cells/kg incohorts of 3, and were eligible for additional infusions at a one loghigher dose after 28 days if there was no evidence of GVHD or theCD4 count was less than the target of 200 cells/l. The initial dosewas escalated by one log in subsequent cohorts if no complicationswere observed in the previous cohort. All patients had pre-DLICD4 counts of 5.0 cells/l, and received 1 to 3 infusions postPCT. Since ATG was used during PCT cytoablation, the first DLIinfusions were administered after ATG levels were 2 mcg/ml.After DLI #1, 4 of 5 matched sibling recipients and 1 of 1 unrelateddonor recipients demonstrated increased CD4 counts with amedian increase of 68 donor derived CD4 cells/l (range 57-460). Three of these patients achieved a CD4 count above 200after 1-3 total infusions. In contrast, none of the haploidenticalrecipients (receiving the smallest doses) had an increase in theirCD4 counts. Two patients developed mild GVHD (1 haploiden-tical Grade II, 1 matched sibling Grade I). In recovering patients,spectratype analysis of CD4 cells comparing donor lymphocytesbefore LLME treatment with lymphocytes recovered from thepatients post DLI demonstrated that 94% of the resolvable Vßfamilies were equally complex. Perforin positive cells, as well asadenovirus and CMV specific lymphocytes, also recovered postPCT in these patients. These preliminary results suggest thatLLME treated DLI can accelerate CD4 reconstitution withoutcausing severe GVHD.45NON-GVHD-INDUCING CD62L- MEMORY T CELLS PROMOTE NEW TCELL REGENERATION FROM HEMATOPOIETIC STEM CELLSChen, B.J., Cui, X., Son, J., Chao, N.J. Duke University MedicalCenter, Durham, NCWe previously demonstrated that allogeneic CD62L- memory Tcells from unprimed donor mice are unable to induce graft-versus-host disease (GVHD) in allogeneic host (Blood. PrepublishedOctober 9, 2003; DOI 10.1182/blood-2003-08-2987). In this pub-lished study, we also demonstrated that in contrast to bulk T cellsand CD62L T cells, CD62L- T cells contribute to post-trans-plant T cell reconstitution not only by peripheral expansion butalso by promoting new T cells from T cell-depleted bone marrow.To investigate whether the new T cells were indeed generatedfrom hematopoietic stem cells, we transplanted 1x106 CD62L-splenic T cells from C57BL/6 mice (H2b, CD45.2) along with5000 purified c-KitThy1.1lowLin-/lowSca-1 hematopoietic stemcells from congenic C57BL/6 mice (H2b, CD45.1) into lethallyirradiated (8.5 Gy) BALB/c mice (H2d, CD45.2). Phenotypic Tcell recovery was then followed weekly in the first 8 weeks and thenonce every other week until day 100 after transplantation. In thismodel, the origin of different T cells can be distinguished by flowcytometry (donor CD62L- T cell-derived: H2DbCD45.2, do-nor stem cell-derived: H2DbCD45.1, host-type: H2Db-CD45.2-). Stem cell-derived T cells could not be detected inperipheral blood in any recipient during the first month followingtransplantation. By day 35, stem cell-derived CD4 and CD8 Tcells became detectable in mice receiving only purified stem cells.However, the addition of CD62L- cells into the graft dramaticallyincreased stem cell-derived CD4 and CD8 T cell numbers inperipheral blood (Table). This effect lasted through day 49. Byday 56, stem cell-derived T cell numbers became equivalent inboth groups. These data demonstrate that CD62L- T cells canaccelerate new T cell development from hematopoietic stem cellspresumably through thymopoiesis. Mature T cells could not me-diate this effect in syngeneic hosts, suggesting that this is anallogeneic effect. CD62L- T cells may be mediating the effectthrough enhancing the level or speed of stem cell engraftmentsince CD62L- T cells also imporved total white cell recovery. Inconclusion, our data suggest that CD62L- memory T cells repre-sent a unique population of T cells which do not cause GVHD butare capable of promoting new donor T cell generation from he-matopoietic stem cells after allogeneic hematopoietic cell trans-plantation.46T CELL DEPLETED HOSTS ARE UNIQUELY SUSCEPTIBLE TO TOLER-ANCE INDUCED BY NON-PROFESSIONAL APCS AND MEDIATED BYIL-10 PRODUCING CD8 T CELLSFry, T.J.1, Melchionda, F.2, Mackall, C.L.1 1. Pediatric OncologyBranch, NCI,NIH, Bethesda, MD; 2. S. Orsola Hospital, University ofBologna, Pediatric Oncology-Hematology & Cell Therapy Unit, Bologna,ItalyBackground: Emerging evidence indicates that the immunobi-ology of T cell depleted (TCD) hosts differs from normal hosts.We observed increased susceptibility to tolerance after T celldepletion and hypothesize that mechanisms at work in this modelare relevant for induction of immune responses following BMT.Methods: Thymectomized, TCD C57BL/6 female mice reconsti-tuted with naive T cells were immunized with male dendritic cells(DC) or male splenocytes. T cell responses to HY were measuredusing skin graft rejection, tetramer and ELISPOT. Male skin graftrejection correlated with expansion of HY-reactive CD8 cells andIFN production in response to HY by CD4 and CD8 cells.Results: TCD recipients of male DC generate HY specific immu-nity but not recipients of male splenocytes. TCD, male splenocyteimmunized animals are tolerant to HY since attempts to sensitizewith male DC were unsuccessful. Splenic B cells were necessaryand sufficient to induce this tolerance. B cell induced tolerance toHY in TCD hosts could not be reversed by restoring normal T cellnumber through placement of a thymic graft or adoptive transferof new naive T cells indicating immune suppression. CD8 T cells,but not CD4 T cells, were sufficient to mediate suppressive toler-ance to HY. T cells from IL10-/- mice could not mediate tolerancewhereas TGF	 neutralization had no effect. Thus, residual CD8cells induce suppressive tolerance to HY after encounter with maleB cells. Since TCD hosts have increased IL7, we hypothesized thatIL7 might play a role in T cell depletion-induced tolerance. In-deed, neutralization of IL7 and IL7R at the time of T-B cellencounter prevented tolerance in the majority of animals. Con-clusions: Although IL7 induced homeostatic expansion in TCDhosts is now widely appreciated as a mechanism which enhancesimmune responses to low affinity and self antigens, the factorswhich prevent autoimmunity in this setting remain unclear. Theseresults give rise to a model wherein increased IL7 in TCD hostsprevents autoimmunity by inducing CD8/IL10 mediated suppres-sive tolerance to antigen presented on non-professional APCs.Therefore TCD hosts are uniquely susceptible to tolerance induc-tion during T cell regeneration due to stringent APC require-ments. While this may prevent autoimmunity during T cell regen-eration, it may also limit the success of immune based therapiesdirected toward infectious and neoplastic antigens in clinical situ-ations involving T cell depletion.Table. Allogeneic CD62L T Cells Promote New T-cellRegeneration from Hematopoietic Stem CellsGroupsCD62LT-cell–derived(cells/LBlood)Stemcell–derived(cells/LBlood)CD4 CD8 CD4 CD8Stem cellonly 3  1 1  1 104  84 18  17stem cell CD62LT cell 32  18* 50  8* 390  141* 97  24**P .01, compared with stem cell only. Data were from day 35.Oral Presentations23BB&MT

T cell depleted hosts are uniquely susceptible to tolerance induced by non-professional APCs and mediated by IL-10 producing CD8+ T cells

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T cell depleted hosts are uniquely susceptible to tolerance induced by non-professional APCs and mediated by IL-10 producing CD8+ T cells 

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T cell depleted hosts are uniquely susceptible to tolerance induced by non-professional APCs and mediated by IL-10 producing CD8+ T cells

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