ALBERT

Description: Regulatory T cells (Treg) play a key role in controlling immune responses following allogeneic hematopoietic stem cell transplantation (HSCT). In murine models, infusion of purified CD4+CD25+ Treg at the time of transplant is sufficient to prevent acute GVHD. In humans, development of acute as well as chronic GVHD has been associated with reduced numbers of Treg following allogeneic HSCT, suggesting that defective reconstitution of this functional cell type can contribute to exacerbation of alloimmune responses. Based on these results, adoptive cellular therapy using purified and in vitro expanded populations of Treg has been proposed as a therapeutic strategy to correct chronic GVHD. Treg are mainly characterized by the constitutive expression of the IL-2 receptor ? chain, CD25 and proliferate in response to IL-2 in vitro. In vivo, the effects of IL-2 on Treg populations are unknown. To examine this question we quantified changes in Treg in 9 patients with CML who previously received low dose IL-2 following allogeneic HSCT. Patients enrolled in this protocol received a daily intravenous infusion of 2 X 105 U IL-2/m2 for 3 months, starting 3 months after CD6 depleted allogeneic bone marrow transplantation (BMT). No patient developed GVHD following IL-2 administration and overall toxicity was minimal. The predominant immunologic effect of IL-2 reported in the initial study was a marked increase in NK cell populations characterized as CD3-CD16+CD56+ as well as total CD56+ cells. In this retrospective analysis we investigated populations of CD4+CD25+ T cells before and 1 to 2 months after the beginning IL-2 treatment. Using RNA extracted from patient PBMC we also assessed the level of expression of the specific transcription factor FOXP3 by quantitative PCR as an alternate marker of Treg in vivo. As initially reported, all 9 patients showed a marked increase in CD3-CD56+ cells 1 to 2 months post IL-2 administration. In contrast, the percent of CD3+ T cells were either unchanged or slightly decreased. The percent of CD4+CD25+ cells within the CD3+ T cell population increased during IL-2 treatment (median: 5.8 pre IL-2 vs 7.6 post IL-2, p-value=0.02). Likewise, FOXP3 expression in the CD3+ population showed 5 to 19 fold increase in 8 of 9 patients during this period (median: 3817 AU pre IL-2 vs. 18924 AU post IL-2, p-value=0.055). These results indicate that administration of low dose IL-2 can augment Treg cells in vivo as reflected by increased ratio of CD4+CD25+/CD3+ T cells as well as higher levels of FOXP3 expression. These studies suggest that prolonged treatment with low dose IL-2 can effectively expand CD4+CD25+ Treg in vivo. This represents a novel strategy for expanding regulatory T cells in vivo and may be useful alone or in conjunction with adoptive cellular therapy with Treg.

Description: Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HSCT). Advances in molecular typing of major histocompatibility complex (MHC) alleles have decreased but not eliminated the occurrence of acute GVHD. Minor histocompatibility antigen (mHA) disparities between patients and their molecularly HLA-identical donors are likely to represent the residual targets of donor immunity. The majority of mHAs are encoded by SNPs which are disparate between patient and donor. Identification of these immunogenic SNPs would provide both a clinical benefit as well as insight into the biology of GVHD. We used Affymetrix high-density arrays to assess 10,043 SNPs spanning the entire genome. We have examined genomic DNA from 36 patient/donor pairs (21 with acute GVHD grades II-IV and 15 without GVHD). All patients and donors were HLA and sex-matched siblings and all patients received T-cell depleted transplants. T cell depletion is important in this context as it removes the potential pharmacogenomic confounding factor of differential sensitivity to immunosuppressive agents. Because the donors and recipients are related, large areas of the genome are identical by descent. Similarly sized blocks of chromosome are disparately inherited. We hypothesized that genomic regions important to the immunopathogenesis of GVHD would be more likely to be disparately inherited in pairs with GVHD than in pairs without GVHD. As a control of our method we compared the area of chromosome 6 surrounding the MHC complex and demonstrated that there was minimal SNP disparity in either GVHD pairs or asymptomatic pairs. We also focused our attention on the region of chromosome 6 outside the MHC area, hypothesizing that disparities in this area would have to result from recombination events. Interestingly the only major area of disparity on Chr 6 was in the gene GMDS, which has been proposed to play a role in the extravasation of activated lymphocytes. We then examined other chromosomes for evidence of genomic regions that that were selectively disparate in GVHD pairs. We were able to identify several genomic regions that appeared to be associated with this outcome. The Lander-Green algorithm was used to estimate the allele sharing between the siblings for each SNP marker and then the disparity score was defined as the average allele sharing of the GVHD group - average allele sharing of the asymptomatic group. The five genomic regions with the highest disparity scores (ranked in order of score) and the gene closest to the region are: 11q14.3 (gene cysteine and histidine-rich domain (CHORD)-containing, zinc binding protein 1), 9p22.2 (gene SH3GL2), 5p15 (gene KIAA0947), 10p15.1 (gene aldo-keto reductase family 1, member C4), and 9p21.2 (Chr 9 ORF 72). The highly disparate regions ranged between 70 and 2200 kb in size. Interestingly the total number of disparate SNPs was not different between pairs with GVHD and pairs without GVHD, supporting the hypothesis that a limited number of SNPs are important immunologic targets. This technique of genome-wide disparity analysis is a promising addition to our ability to define important mHA.

Description: Previous trials of allogeneic bone marrow transplantation (BMT) in patients with multiple myeloma (MM) have demonstrated high response rates but also high transplantation-related mortality (TRM) and high relapse rates. Exploitation of this strategy remains of interest because donor lymphocyte infusions (DLIs) can induce a potent graft-versus-myeloma (GVM) effect. CD6 T-cell–depleted allogeneic BMT was combined with prophylactic CD4+ DLI administered 6 to 9 months after BMT in an effort to reduce TRM and to induce a GVM response after BMT. Twenty-four patients with matched sibling donors and chemotherapy-sensitive disease underwent BMT. CD6 T-cell depletion of donor bone marrow was the sole method of graft-versus-host disease (GVHD) prophylaxis. GVHD after BMT was minimal, 1 (4%) grade III and 4 (17%) grade II GVHD. Fourteen patients received DLI, 3 in complete response and 11 with persistent disease after BMT. Significant GVM responses were noted after DLI in 10 patients with persistent disease, resulting in 6 complete responses and 4 partial responses. After DLI, 50% of patients developed acute (≥ II) or extensive chronic GVHD. Two-year estimated overall survival and current progression-free survival (PFS) for all 24 patients is 55% and 42%, respectively. The 14 patients receiving DLI had an improved 2-year current PFS (65%) when compared with a historical cohort of MM patients who underwent CD6-depleted BMT survived 6 months with no GVHD and did not receive DLI (41%) (P = .13). Although this study suggests that prophylactic DLI induces significant GVM responses after allogeneic BMT, only 58% of patients were able to receive DLI despite T-cell–depleted BMT. Therefore, less toxic transplantation strategies are needed to allow a higher proportion of patients to receive DLI and the benefit from the GVM effect after transplantation.

Description: Following myeloablative therapy, it is unknown to what extent age-dependent thymic involution limits the generation of new T cells with a diverse repertoire. Normal T-cell receptor gene rearrangement in T-cell progenitors results in the generation of T-cell receptor rearrangement excision circles (TRECs). In this study, a quantitative assay for TRECs was used to measure T-cell neogenesis in adult patients with leukemia who received myeloablative therapy followed by transplantation of allogeneic hematopoietic stem cells. Although phenotypically mature T cells had recovered by 1 to 2 months after bone marrow transplantation (BMT), TREC levels remained low for 3 months after BMT. T-cell neogenesis became evident by 6 months, and normal levels of adult thymic function were restored at 6 to 12 months after BMT. Subsequent leukemia relapse in some patients was associated with reduced TREC levels, but infusion of mature donor CD4+ T cells resulted in rapid restoration of thymic function. These studies demonstrate that T-cell neogenesis contributes to immune reconstitution in adult patients and suggest that thymic function can be manipulated in vivo.

Description: H-Y minor histocompatibility antigens (mHA) are common targets of immune responses following allogeneic hematopoietic stem cell transplantation (HSCT) in male patients who receive stem cells from female donors. These H-Y antigens are encoded by a group of genes located on the non-recombining portion of the Y chromosome. H-Y genes are ubiquitously expressed with 1 to 13% disparity at the protein level with their X homologues. The same portion of the Y chromosome also contains a distinct group of 11 Y-specific genes, for which there are no X homologues. Expression of Y-specific genes is reportedly restricted to the testis thereby limiting the potential relevance of these proteins as immune targets following allogeneic HSCT. However, atypical expression of Y-specific genes has recently been reported in prostate cancer, suggesting that these genes might follow a pattern of expression characteristic of cancer-testis antigens. In this study, we investigated the expression of a representative Y-specific gene, PRY, in male leukemia cell lines, and examined the immunogenicity of this protein in male patients who received allogeneic HSCT from female donors. Using DNAse treated RNA in RT-PCR experiments we showed that PRY gene is expressed in 3 of 6 male leukemia cell lines tested but not in 4 female cell lines. Results were confirmed by southern blotting of PCR products using an internal specific probe. We next assessed PRY expression in normal blood cells collected from 3 male and 3 female donors. In contrast to what has previously been reported, PRY gene was found expressed at low levels in blood cells from all male donors but not from female donors. Although the precise phenotype of cells expressing PRY remains unknown, expression in normal hematopoietic as well as tumor cells suggested that Y-specific gene products could also elicit immune responses after sex mismatched allogeneic HSCT. We used a series of overlapping peptides encompassing the entire sequence of PRY in ELISA assays to examine the antibody response to PRY antigen in male recipients of female transplant. Thus far, 1 of 13 serum samples has been positive for antibody to PRY. This sample, collected approximately one year post-transplant, strongly reacted with a single peptide, indicating that the patient had developed a B cell response to the PRY antigen. This patient with AML had received a HSCT from his HLA identical female sibling. After transplant, he developed acute and chronic GVHD. Remarkably, this patient also developed a strong CD4+ T cell and B cell response to another H-Y antigen, DBY, following HSCT. Studies are ongoing to further characterize the immune response elicited towards this newly defined antigen. Overall, our data indicate that the Y-specific gene PRY is expressed in normal blood cells as well as leukemia cells. Following allogeneic HSCT, PRY can also trigger B cell immunity in male patients with female donors. Further studies are required to determine whether other Y-specific gene products are also immunogenic and constitute a new category of mHA with significant implications in the development of GVL and GVH reactions.

Description: Reconstitution of T-cell immunity after bone marrow transplantation (BMT) is often delayed, resulting in a prolonged period of immunodeficiency. Donor lymphocyte infusion (DLI) has been used to enhance graft-versus-leukemia activity after BMT, but the effects of DLI on immune reconstitution have not been established. We studied 9 patients with multiple myeloma who received myeloablative therapy and T-cell–depleted allogeneic BMT followed 6 months later by infusion of lymphocytes from the same donor. DLI consisted of 3 × 107 CD4+ donor T cells per kilogram obtained after in vitro depletion of CD8+ cells. Cell surface phenotype of peripheral lymphocytes, T-cell receptor (TCR) Vβ repertoire, TCR rearrangement excision circles (TRECs), and hematopoietic chimerism were studied in the first 6 months after BMT and for 1 year after DLI. These studies were also performed in 7 patients who received similar myeloablative therapy and BMT but without DLI. Phenotypic reconstitution of T and natural killer cells was similar in both groups, but patients who received CD4+ DLI developed increased numbers of CD20+ B cells. TCR Vβ repertoire complexity was decreased at 3 and 6 months after BMT but improved more rapidly in patients who received DLI (P = .01). CD4+ DLI was also associated with increased numbers of TRECs in CD3+ T cells (P