Donor leukocyte infusions induce remissions in some patients (pts) with hematologic malignancies who relapse after allogeneic hematopoietic cell transplantation (HCT). However, graft vs host disease (GVHD) remains the major complication of this strategy. Cytokine induced killer (CIK) cells are a unique population of cytotoxic T lymphocytes that express the CD3+CD56+ phenotype and show marked upregulation of the NK cell receptor, NKG2D (CD314). CIK cells are non-MHC restricted, and NKG2D dependent in target recognition and cytotoxicity. We explored the feasibility of ex vivo expansion of allogeneic CIK cells for pts with relapsed hematologic malignancies after allogeneic HCT. Eighteen patients with a median age of 53 years (range 20–69) received CIK cell infusions based on CD3+cells/kg at escalating doses of 1×107 (n=4), 5×107 (n=6) and 1×108 (n=8). The median expansion of CD3+ cells was 12 fold (range 4–91 fold). CD3+CD56+ cells represented a median of 11% (range 4–44%) of the harvested cells with a median 31 fold (range, 7–515 fold) expansion. Median CD3+CD314+ expression was 53% (range, 32–78%) of harvested cells. Significant cytotoxicity was demonstrated in vitro against a panel of human tumor cell lines. Acute GVHD, grades I–II, were seen in 2 patients and 1 patient has limited chronic GVHD. After a median followup of 20 months (range 1–69 months) from CIK infusion, the median overall survival was 28 months and median event free survival was 4 months. All deaths were due to relapsed disease, however, 5 patients had longer remissions after infusion of CIK cells than from allogeneic transplantation to relapse. This form of adoptive immunotherapy is well tolerated and induces a low incidence of GVHD supporting further investigation as an upfront modality to enhance GVT responses in high risk patient populations.

Preclinical studies have shown that persistent mixed chimerism is linked to acceptance of organ allografts without immunosuppressive (IS) drugs. Mixed chimerism refers to continued mixing of donor and recipient hematopoietic cells in recipient tissues after transplantation of donor cells. To determine whether persistent mixed chimerism and tolerance can be established in patients undergoing living donor kidney transplantation, we infused allograft recipients with donor T cells and hematopoietic progenitors after posttransplant lymphoid irradiation. In 24 of 29 fully human leukocyte antigen (HLA)–matched patients who had persistent mixed chimerism for at least 6 months, complete IS drug withdrawal was achieved without subsequent evidence of rejection for at least 2 years. In 10 of 22 HLA haplotype–matched patients with persistent mixed chimerism for at least 12 months, reduction of IS drugs to tacrolimus monotherapy was achieved. Withdrawal of tacrolimus during the second year resulted in loss of detectable chimerism and subsequent rejection episodes, unless tacrolimus therapy was reinstituted. Posttransplant immune reconstitution of naïve B cells and B cell precursors was more rapid than the reconstitution of naïve T cells and thymic T cell precursors. Robust chimerism was observed only among naïve T and B cells but not among memory T cells. No evidence of rejection was observed in all surveillance graft biopsies obtained from mixed chimeric patients withdrawn from IS drugs, and none developed graft-versus-host disease. In conclusion, persistent mixed chimerism established in fully HLA- or haplotype-matched patients allowed for complete or partial IS drug withdrawal without rejection.

V(D)J recombination in lymphoid cells is a site-specific process in which the activity of the recombinase enzyme is targeted to signal sequences flanking the coding elements of antigen receptor genes. The order of the steps in this reaction and their mechanistic interdependence are important to the understanding of how the reaction fails and thereby contributes to genomic instability in lymphoid cells. The products of the normal reaction are recombinant joints linking the coding sequences of the receptor genes and, reciprocally, the signal ends. Extrachromosomal substrate molecules were modified to inhibit the physical synapsis of the recombination signals. In this way, it has been possible to assess how inhibiting the formation of one joint affects the resolution efficiency of the other. Our results indicate that signal joint and coding joint formation are resolved independently in that they can be uncoupled from each other. We also find that signal synapsis is critical for the generation of recombinant products, which greatly restricts the degree of potential single-site cutting that might otherwise occur in the genome. Finally, inversion substrates manifest synaptic inhibition at much longer distances than do deletion substrates, suggesting that a parallel rather than an antiparallel alignment of the signals is required during synapsis. These observations are important for understanding the interaction of V(D)J signals with the recombinase. Moreover, the role of signal synapsis in regulating recombinase activity has significant implications for genome stability regarding the frequency of recombinase-mediated chromosomal translocations.The exons encoding the immunoglobulin and T-cell receptor variable region domains are assembled during lymphocyte ontogeny by V(D)J recombination (reviewed in references 24 and 34). This site-specific reaction is directed by a pair of joining signals recognized by components of the recombination activity. The consensus sequence of each signal consists of a palindromic heptamer and an AST-rich nonamer. These two elements are separated by a 12-base spacer at one signal and a 23-base spacer at the other. The recombination crossover point for each signal is at the end of the heptamer on the side distal from the nonamer.During recombination, coding ends and signal ends are generated by recombinase-mediated cleavage at the crossover sites. The two coding ends are joined to form what is termed a coding joint, and the two signal ends are joined to form a signal joint. Modification of the coding ends occurs by terminal deoxynucleotide transferase-associated nucleotide addition (7,19,29), by P-element addition (18,29), and by nucleolytic chewback of the exposed ends prior to joint resolution (reviewed in references 21 and 24). These modifications are important to the diversification of the antigenbinding repertoire.Elucidating the order of the steps in this reaction and their mechanistic interdependence is important to our understanding of how the reaction fails and thereby contributes to...

The influence of graft composition on clinical outcomes after reduced-intensity conditioning is not well-characterized. In this report we prospectively enumerated CD34 ؉ , CD3 ؉ , CD4 ؉ , and CD8 ؉ cell doses in granulocyte colony-stimulating factormobilized peripheral blood mononuclear cell (