Antithymocytes globulins: Time to revisit its use in kidney transplantation?

T cell depletion by polyclonal antithymocyte globulins (ATG) has been used for many years in both organ and hematopoietic cell transplantation as an induction and rejection therapy. Nevertheless, its use remains largely empirical and many clinical questions, such as the determination of an individualized dose, therapeutic relevance of nondepletive effects, or prediction of long-term effects, are still unresolved. This review evaluates the evidence-based knowledge and the uncertainties concerning ATG, and suggests perspectives and opportunities for modern use of this old drug.

Setting the standard in T-cell-depleted haploidentical transplantation and beyond.

Much progress has been made in the clinical, biological and technical aspects of the T-cell-depleted haploidentical hematopoietic stem cell transplantation (HSCT). Our experience demonstrates that infusing a megadose of extensively T-cell-depleted hematopoietic peripheral blood stem cells after an immuno-myeloablative conditioning regimen in acute leukemia patients ensures sustained engraftment with minimal GvHD without the need of any post-transplant immunosuppressive treatment. Since our first successful pilot study, our efforts have concentrated on developing new conditioning regimens, optimizing the graft processing and improving the post-transplant immunological recovery. The results we have so far achieved in high risk acute leukemia patients show that haploidentical transplantation is now a clinical reality. The present challenge is to reduce post-transplant infectious mortality and several strategies are being tested.

A novel high throughput immunomagnetic cell sorting system for potential clinical scale depletion of T cells for allogeneic stem cell transplantation.

OBJECTIVE: To develop an immunomagnetic cell separation system for allogeneic hematopoietic stem cell (HSC) transplantations, which can achieve a high level of T-cell depletion (at least 4.0 log(10)), high level of recovery of hematopoietic stem cells (>90%), with a high throughput (>10(6) cells/second). METHODS: Peripheral blood leukocytes (PBLs) from buffy coats were spiked with CD34-expressing cells (KG1a) to mimic a leukaphoresis product containing stimulated HSCs. T cells were labeled with anti-CD3(+) Dynabeads and separated in a quadrupole magnetic cell sorter (QMS). The performance of the system with respect to T-cell depletion and recovery of non-T cells and spiked KG1a was determined using four-color, flow cytometry analysis, with the aid of Trucount cell-concentration calibration beads. Limiting dilution assays were also performed to quantify the log(10) depletion of clonable T cells. RESULTS: While the general performance of the QMS system is governed by proven theoretical principles, significant system variability exist, not all of which can be explained by our current understanding. Consequently, a factorial design was employed, guided by JMP software, to optimize the labeling conditions and operation of the QMS focused on maximizing the depletion of T cell, and recovery of unlabeled cells including KG1a cells. From these studies, an optimized, no wash, immunomagnetic labeling protocol and optimized QMS operating conditions were developed. For an average initial cell concentration of 1.7 x 10(8) total cells, an average 3.96 +/- 0.33 log(10) depletion (range, 3.53-4.34) of CD3(+)CD45(+) cells with a mean 99.43% +/- 4.23% recovery of CD34(+)CD45(+) cells (range, 94.38-104.90%) was achieved at a sorting speed of 10(6) cells/s (n = 6). Limiting dilution assays on the T-cell depleted fractions, which gave a log(10) depletion of 3.51 for the clonable T cells. CONCLUSION: We suggest that this system will provide superior performance with respect to T-cell depletion and CD34(+) recovery for clinical allogeneic bone marrow transplants. Ongoing studies, on a clinical scale, are being conducted to demonstrate this claim.

T-cell reconstitution after unmanipulated, CD8-depleted or CD34-selected nonmyeloablative peripheral blood stem-cell transplantation.

BACKGROUND: We have previously shown that CD8 depletion or CD34 selection of peripheral blood stem cells (PBSC) reduced the incidence of acute graft-versus-host disease (GvHD) after nonmyeloablative stem-cell transplantation (NMSCT). In this study, we analyze the effect of CD8 depletion or CD34 selection of the graft on early T-cell reconstitution. METHODS: Nonmyeloablative conditioning regimen consisted in 2 Gy total-body irradiation (TBI) alone, 2 Gy TBI and fludarabine, or cyclophosphamide and fludarabine. Patients 1 to 18 received unmanipulated PBSC, patients 19 to 29 CD8-depleted PBSC, and patients 30 to 35 CD34-selected PBSC. RESULTS: T-cell counts, and particularly CD4+ and CD4CD45RA+ counts, remained low the first 6 months after nonmyeloablative stem-cell transplantation (NMSCT) in all patients. CD34 selection (P<0.0001) but not CD8 depletion of PBSC significantly decreased T-cell chimerism. Donor T-cell count was similar in unmanipulated compared with CD8-depleted PBSC recipients but was significantly lower in CD34-selected PBSC recipients (P=0.0012). T cells of recipient origin remained stable over time in unmanipulated and CD8-depleted PBSC patients but expanded in some CD34-selected PBSC recipients between day 28 and 100 after transplant. Moreover, whereas CD8 depletion only decreased CD8+ counts (P<0.047), CD34 selection reduced CD3+(P<0.001), CD8+(P<0.016), CD4+ (P<0.001), and CD4+CD45RA+ (P<0.001) cell counts. T-cell repertoire was restricted in all patients on day 100 after hematopoietic stem-cell transplantation but was even more limited after CD34 selection (P=0.002). CONCLUSIONS: Despite of the persistence of a significant number of T cells of recipient origin, T-cell counts were low the first 6 months after NMSCT. Moreover, contrary with CD8 depletion of the graft that only affects CD8+ lymphocyte counts, CD34 selection dramatically decreased both CD8 and CD4 counts.

T-cell--depleted allogeneic bone marrow transplantation followed by donor lymphocyte infusion in patients with multiple myeloma: induction of graft-versus-myeloma effect.

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 (> or = 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. (Blood. 2001;98:934-939)

Prevention of acute graft-versus-host-disease by selective depletion of T cells reactive with minor histocompatibility antigens on epithelial cells.

Graft-versus-host disease (GVHD) is a major obstacle in allogeneic hematopoietic stem cell transplantation (HSCT). Mature donor T-cells present in the graft play a pivotal role in the development of acute GVHD. On the other hand, mature donor T-cells in the graft are also crucial for the elimination of residual tumor cells still present in the patient after HSCT. Whether donor T cells act non-specifically against the patient, including an overlapping GVHD/GVL reactivity, or some donor T cells have GVHD reactivity while other donor T cells have GVL reactivity is still unclear. Some in-vitro data are suggestive that selective T cell depletion techniques are possible by which GVHD-reactive T cells can be eliminated while GVL-reactive T cells are preserved. Here we update some approaches of selective T cell depletion that have been developed in our laboratory.

The number of donor CD3(+) cells is the most important factor for graft failure after allogeneic transplantation of CD34(+) selected cells from peripheral blood from HLA-identical siblings.

This study analyzed the characteristics of 257 HLA-identical sibling transplants of granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells depleted of T cells by CD34(+) positive selection (allo-PBT/CD34(+)) for their effect on the incidence of graft failure. Twenty-four patients developed graft failure (actuarial probability, 11%; 95% confidence interval, 7.1-14. 9). Prognostic factors considered were sex and age of donor and recipient, donor-recipient blood group compatibility, diagnosis, disease status at transplant, conditioning regimen, cytomegalovirus serology, number of CD34(+) and CD3(+) cells infused, and cryopreservation. The major factor associated with graft failure was the number of CD3(+) cells in the inoculum. Twenty-three of 155 patients receiving a T-cell dose in the graft less than or equal to 0.2 x 10(6)/kg experienced graft failure, compared with only one of 102 patients receiving more than 0.2 x 10(6)/kg (actuarial probability 18% vs 1%, respectively; P =.0001). The actuarial probability of graft failure progressively increased as the number of CD3(+) cells in the graft decreased, which was determined by grouping the number of CD3(+) cells in quartiles (log-rank P =.03; log-rank for trend P =.003). In the multivariate analysis by the proportional hazard method, 2 covariates entered into regression at a significant level: CD3(+) cells less than or equal to 0.2 x 10(6)/kg (risk ratio = 17; P <.0001), and patients with chronic myelogenous leukemia (CML) conditioned with busulphan-based regimens (risk ratio = 4.8; P =.001). From these results it appears that the number of CD3(+) cells in the inoculum-with a threshold of 0.2 x 10(6)/kg or less-is the most critical factor in maintaining a sustained engraftment in allo-PBT/CD34(+) from HLA-identical siblings. In addition, for patients with CML receiving 0.2 x 10(6)/kg or less CD3(+) cells, total body irradiation might be better than busulphan-based regimens.

Monitoring universal leucodepletion performance: the current issues within components production and testing.

The UK NBS completed the introduction of universal leucodepletion in November 1999. This involved the implementation of new methods of production, testing and monitoring, the development of which is still ongoing. Whilst important lessons have been learnt, much remains to be achieved in respect of quality improvement through standardisation of procedures, processes and product modifications to improve safety/efficacy of therapeutic blood components. Since the introduction of universal leucodepletion, many changes in component production and testing have occurred. The primary goal of this report is to review the level of standardisation and harmonisation currently achieved for low leucocyte counting within the NBS and discuss remedial actions undertaken to improve the standard of blood component production and quality monitoring and to highlight some of the related issues which are currently being worked on. The following issues are discussed.

[Leukocyte depletion]. / La déleucocytation.

Leukodepletion is a significant factor of improvement of the quality and safety of blood cell components. By reducing residual leukocytes in the products to a rate below 1 million per unit, the risks of transmitting intralymphocyte viruses, provoking shivers and hypothermia reactions, and inducing anti-HLA alloimmunization are diminished, as well as bacterial contamination risks. The constant improvement of the performance and the use of leukodepletion filters allows this technique to be generalized to all red blood cell and platelet concentrates. Leukocytes retention devices on these filters are numerous and sensitive to the conditions for material use. Implementation of Leukodepletion at production stage must be carried out in a mastered framework of quality assurance.

Leukocyte depletion of cellular blood components.

Clinical studies have indicated that the use of leukocyte-reduced cellular blood components produced in the laboratory may prevent febrile reactions and delay or prevent alloimmunization to HLA antigens and refractoriness to platelet transfusion. Additional investigations regarding the effects of the use of leukocyte-reduced blood components were reported during the past year. A recent study in patients with hematologic malignancy that employed the commonly used bedside leukocyte-reduction filters failed to confirm a decrease in the rate of alloimmunization, except in a subgroup of patients with acute myelogenous leukemia. Another major multicenter trial confirmed the effectiveness of leukocyte-reduced blood components in the prevention of cytomegalovirus infection. The effect of allogeneic leukocytes in transfused blood on immune function in patients undergoing colorectal surgery continues to receive attention. Whereas one study failed to demonstrate an adverse effect of standard blood components on disease recurrence or survival, a second study demonstrated a marginally significant decrease in infectious complications in patients who received only leukocyte-reduced blood. Increasingly efficient leukocyte-reduction filters have been developed for cellular blood components, many of which are best suited for laboratory filtration of unstored blood. Laboratory studies indicate that prestorage leukocyte-reduction of cellular blood components does not impair erythrocyte or platelet function and will not increase the incidence of microbial contamination of blood. New methods that employ flow cytometry should enable improved quality control of blood components rendered leukocyte-reduced by the newer, more efficient filters. Finally, a cost-benefit analysis suggests that the appropriate use of leukocyte-reduction filters for acute leukemia patients may reduce the cost of health care to these patients.

Selective depletion of bone marrow T lymphocytes with anti-CD5 monoclonal antibodies: effective prophylaxis for graft-versus-host disease in patients with hematologic malignancies.

Seventy-one patients with hematologic malignancies received bone marrow from a histocompatible sibling (n = 48) or a partially matched relative (n = 23) that had been depleted of CD5+ T cells with either an anti-CD5 mooclonal antibody (MoAb) plus complement (anti-Leu1 + C) or an anti-CD5 MoAb conjugated to ricin A chain (ST1 immunotoxin [ST1-IT]). These patients received intensive chemoradiotherapy consisting of cytosine arabinoside, cyclophosphamide, and fractionated total body irradiation. Both anti-Leu1 + C and ST1-IT ex vivo treatments effectively depleted bone marrow of T cells (97% and 95%, respectively). Overall, primary and late graft failure each occurred in 4% of evaluable patients. The diagnosis of myelodysplasia was a significant risk factor for graft failure (P less than .001), and if myelodysplastic patients were excluded, there were no graft failures in major histocompatibility complex (MHC)-matched patients and 2 of 23 (8.7%) in MHC-mismatched patients. The actuarial risk of grade 2 to 4 acute graft-versus-host disease (GVHD) was 23% in MHC-matched patients and 50% in MHC-mismatched patients. In MHC-matched patients, acute GVHD tended to be mild and treatable with corticosteroids. Chronic GVHD was observed in 6 of 36 (17%) MHC-matched patients and none of 11 MHC-mismatched patients. There were no deaths attributable to GVHD in the MHC-matched group. Epstein-Barr virus-associated lymphoproliferative disorders were observed in 3 of 23 MHC-mismatched patients. The actuarial event-free survival was 38% in the MHC-matched patients versus 21% in the MHC-mismatched patients. However, if outcome is analyzed by risk of relapse, low-risk patients had a 62% actuarial survival compared with 11% in high-risk patients. These data indicate that the use of anti-CD5 MoAbs can effectively control GVHD in histocompatible patients, and that additional strategies are required in MHC-mismatched and high-risk patients.