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0Review
Immune Reconstitution after Haploidentical Hematopoietic Stem Cell Transplantation
Ying-Jun Chang1, Xiao-Yu Zhao1, Xiao-Jun Huang1,2 *
1 Peking University People's Hospital and Peking University Institute of Hematology, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Beijing, China
2 Peking-Tsinghua Center for Life Sciences, Beijing, China
American Society for Blood and Marrow Transplantation
Article history: Received 7 August 2013 Accepted 29 November 2013
Key Words:
Haploidentical transplantation Immune reconstitution Natural killer cell T cell
ABSTRACT
Haploidentical hematopoietic stem cell transplantation (HSCT) offers the benefits of rapid and nearly universal donor availability and has been accepted worldwide as an alternative treatment for patients with hematologic malignancies who do not have a completely HLA-matched sibling or who require urgent transplantation. Unfortunately, serious infections and leukemia relapse resulting from slow immune reconstitution remain the 2 most frequent causes of mortality in patients undergoing haploidentical HSCT, particularly in those receiving extensively T cell—depleted megadose CD34+ allografts. This review summarizes advances in immune recovery after haploidentical HSCT, focusing on the immune subsets likely to have the greatest impact on clinical outcomes. The progress made in accelerating immune reconstitution using different strategies after haploidentical HSCT is also discussed. It is our belief that a predictive immune subset—guided strategy to improve immune recovery might represent a future clinical direction.
© 2014 American Society for Blood and Marrow Transplantation.
INTRODUCTION
Haploidentical hematopoietic stem cell (HSC) transplantation (HSCT) is available for nearly all patients and has no search or acquisition costs [1-9]. Over the past decade, many haploidentical transplantation protocols, including T cell—replete and T cell—depleted (TCD) haplotype HSCT, depending on whether or not allografts have been engineered in vitro, have demonstrated promising clinical outcomes [4,8,9]. Unfortunately, serious infections and disease relapse resulting from delayed immune reconstitution remain the 2 most frequent causes of mortality after allo-geneic HSCT, particularly in patients who received extensively TCD CD34+ cell megadose allografts [10-15]. Advances in the understanding of immune recovery profiles in haploidentical recipients [8,16-47], along with new methods of modifying donor T cells [48-50] and natural killer (NK) cells [51], have made it possible to establish new strategies to improve post-transplantation immunologic reconstitution [9,52-58].
In this review, we summarize advances in immune recovery after haploidentical HSCT, focusing on the recovered immune subsets likely to have the greatest impact on clinical outcomes [18,23,33,42,45,59-63]. We compare the differences in immune reconstitution between hap-loidentical transplantation and other transplantation strategies, including HLA-identical sibling donor transplantation, HLA-matched unrelated donor transplantation, and umbilical cord blood transplantation. In addition, we discuss recent advances in the enhancement of immune reconstitution after haploidentical HSCT [64-70].
Financial disclosure: See Acknowledgments on page 447.
* Correspondence and reprint requests: Xiao-Jun Huang, MD, Professor, Peking University Institute of Hematology, Peking University People's Hospital, No. 11 Xizhimen South St, Beijing 100044, China. E-mail address: xjhrm@medmail.com.cn (X.-J. Huang).
KINETICS OF IMMUNE RECOVERY AFTER HAPLOIDENTICAL HSCT
Different immune cell subgroups recover at different rates after haploidentical HSCT (Table 1 ). The conditioning regimen is followed by a "neutropenic" phase that lasts until neutrophils reconstitute, at a median of approximately 11 to 12 days after TCD HSCT with high doses of CD34+ cells [8,38], approximately 15 days after unmanipulated haploidentical steady-state bone marrow (BM) allografts, approximately 21 days after granulocyte colony-stimulating factor (G-CSF)-primed BM allografts, and approximately 13 days after G-CSF—stimulated blood and BM allografts [2,7,71]. The recovery of neutrophil function (eg, chemotaxis, phagocytosis, superoxide production, killing of bacteria) in haploidentical settings remains poorly understood, however.
The rapid recovery of NK cells after haploidentical transplantation is based on an expansion of the cytokine-producing CD56bright NK cell subsets in both T cell—replete and TCD settings [16,17,24,41]. Although the absolute number of overall NK cells usually recovers to the donor's level by day 30 post-HSCT, recovery may be delayed by the development of graft-versus-host disease (GVHD) [8,24]. The function of NK cell recovery is regulated by the balance of activating and inhibitory signals transmitted by different cell surface receptors. Thus, the expression of activating NK receptors (NKRs), such as NKP46, NKP44, NKP30, and NKG2D, as well as inhibiting NKRs, such as CD158a, CD158b, CD158e, and NKG2A, is essential for NK cell activation.
After haploidentical HSCT, the overall expression of activating NKRs and inhibitory NKRs is reduced, whereas CD94/ NKG2A expression is increased. NKG2A recovery is inversely correlated with CD158 recovery in the year after HSCT. This altered phenotype includes more CD56bright cells, fewer CD56dim NK cells, and altered CD94/NKGA expression. Activation or inhibition of NKR expression during early reconstitution is associated with lower levels of in vitro NK
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cytotoxicity after haploidentical HSCT. The reconstitution of killer cell immunoglobulin-like receptors (KIRs) is influenced by many factors, including the conditioning regimen, level of T cell depletion, and the use of immune suppression after transplantation, as recently reviewed by Zhao et al. [72]. Monocyte engraftment is rapid, with normal values on day +15 after unmanipulated haploidentical blood and BM transplantation [25]. In HLA-identical HSCT settings, the absolute monocyte counts at day +30 (>300 cells/pL) are strongly associated with improved survival [73,74], whereas no association between monocyte recovery and outcome after haploidentical HSCT has been reported.
Dendritic cells (DCs) are highly important antigen-presenting cells with central roles in initiating and modulating immune responses. Human peripheral blood DCs are divided into 2 major subsets: myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). mDCs are further subdivided into 2 subsets: T helper cell (Th) 1-promoting mDCs (mDC1s) and Th2-promoting mDCs (mDC2s) [75]. Our preliminary data showed extremely low mDC1, mDC2, and PDC counts at 1-3 months post-HSCT, which recovered to normal values by 1 year after unmanipulated haploidentical blood and BM transplantation [25]. A gradual increase in the DC population after transplantation was also reported in pediatric patients with acute leukemia who received CD3/CD19-depleted grafts from haploidentical donors [41]. The early delayed immune reconstitution of DCs may contribute in part to an increased rate of postengraftment bacterial and fungal infections [76], although little is known about the recovery of DC function after haploidentical transplantation [25,41].
Invariant NK T (iNKT) cells are a specialized subset of T cells that use their T cell receptors (TCRs) to recognize self and foreign lipids presented by CD1d as cognate antigens. These cells can have protective or harmful roles in many pathological states, including microbial infection, autoimmune disease, allergic disease, and cancer [77]. In pediatric patients, de Lalla et al. [33] observed that iNKT recovered slowly after CD34-selected haploidentical HSCT and reached normal reference values by 18 months post-transplantation. Their data also suggest that the frequency of iNKT cells is significantly correlated with the remission state after haploidentical HSCT. In HLA-identical transplantation settings, Chaidos et al. [78] reported that CD4(-) iNKT cell dose was the sole graft parameter predictive of clinically significant acute GVHD. This interesting finding awaits confirmation in haploidentical HSCT settings.
Detailed analyses of adaptive immune reconstitution have been performed by researchers at various transplantation centers [16,17,22,25-27,29,32,33,38,41,45,79-81]. After CD34-selected haplodentical HSCT, mean (±standard deviation) CD4+ cell counts ranged from 100 ± 40/L to 200 ± 20/L at 10 months post-transplantation, and rose thereafter. The mean CD8+ cell count reached 230 ± 80/L on day +60, followed by a steady rise to 570 ± 125/L by day +300. The mean CD16+ NK cell count reached 400/L stably by day +30 [38]. The recovered NK cells may enhance the graft-versus-leukemia effect [38-82]. A German group used negative selection of CD3/CD19 cells to retain NK cells, monocytes, and DCs in haploidentical allografts and found delayed T cell reconstitution, with a median of 369 CD3+ cells/pL, 177 CD4+ cells/pL, and 193 CD8+ cells/pL on day +400 [8,83]. In our unmanipulated haploidentical blood and BM transplantation protocol, a median absolute number of 277 CD4+ T cells/pL and 884 CD8+ T cells/pL were recovered at 1 year post-transplantation [25]. Immune recovery
after unmanipulated haploidentical HSCT appears to be more rapid than CD34-selected haploidentical transplantation; however, the 2 transplantation methods are not strictly comparable [17,25,38,39].
The expansion of memory T cells (especially CD4+ memory T cells, which reconstitute later than CD8+ memory T cells and rely more on thymic production of naive Tcells) results in a significant inversion of the CD4/CD8 ratio up to 1 year after transplantation [25,39,41]. In contrast to most previously reported results [25,27,36,38,39,41], no significant inversion of the CD4/CD8 ratio was observed after haploidentical HSCT using reduced-intensity conditioning and CD3/CD19-depleted grafts [17]. In the TCD haploidentical HSCT protocol [45], the main lymphocyte populations (including CD3+ Tcells, CD4+ Tcells, CD8+ Tcells, and B cells) and the CD4/CD8 ratio reconstituted to age-matched control levels by 4 to 6 years post-transplantation. Similar kinetics in B cell recovery have been reported by our group and others [17,25,41,84], with absolute numbers of B cells increasing gradually and reaching normal levels more than 1 year after HSCT.
Regarding the reconstutiton of T cell function, an interesting finding is that the ability of T cells to secrete IFN and IL-4 recovers to normal level by day +30 post-HSCT in patients without acute GVHD, although TCR rearrangement excision DNA circle (TREC) levels remain low for 12 to 24 months after unmanipulated haploidentical HSCT [29]. At 4 to 6 years after TCD haploidentical HSCT, recipients demonstrate higher proportions of CD31+ naïve CD4+ Tcells compared with donors [17,45]. The signal-joint TCR excision circle (sjTREC) levels in the recipients tend to be higher than the levels in donors, which are similar to those seen in age-matched control subjects.
The foregoing observations suggest that long-term T cell reconstitution is critically influenced by de novo T cell production, reflecting thymopoiesis as a central mechanism. A skewed and restricted distribution of TCR repertoires has been observed at less than 180 days post-transplantation [17]. Azevedo et al. [45] reported that at 4-6 years posttransplantation, patient CD4+ T cells and CD8+ T cells exhibit a diverse T cell repertoire and reconstitute a diverse regulatory T cell (Treg) pool through a mechanism involving de novo thymic production. Their findings provide strong evidence that a normal immune system can be reconstituted after haploidentical HSCT; however, the small number of patients in their study precluded from a subgroup analysis to identify the factors influencing immune reconstitution. In addition, the function of T cells in unmanipulated hap-loidentical HSCT remains to be investigated in depth [2,71,85-88].
More recently, Ciurea et al. [36] demonstrated that compared with patients who underwent TCD haploidentical HSCT, patients who received T cell-replete haploidentical allografts exhibit better immune reconstitution of T cell subsets, including CD4+ Tcells, CD8+ Tcells, naive Tcells, and memory T cells, during the first 6 months after transplantation (Table 1). The authors suggested that improved immune reconstitution might contribute to the improved early outcomes observed with the use ofTcell-replete grafts [36]. Thus, a prospective study to compare immune recovery between TCD and Tcell-replete haploidentical approaches is warranted.
Overall, the first 90 days after haploidentical HSCT are characterized by persistent CD4+ and CD4+ naive T cells, B cell lymphopenia, and low thymic function, which render patients especially susceptible to viral and fungal infections
Immune Reconstitution in Hapolidentical Transplantation and MSDT, MUDT, and Umbilical Cord Blood Transplantation
Federmann et al. [17] Chang et al. [25] Ciurea et al. [36] Pérez-Martmez et al. [41] Kesserwan et al. [43],* Dominietto A et al. [93] *
MSDT HBMT TCD T Cell Replete HIT MUDT ALT CBIBT HIT MSDT
No. of patients 28 25 50 33 32 15 15 21 125 103 40 176
Age, yr, median (range) 45 (19-65) 38.5 (24-57) 24.5 (7-48) 36 (18-56) 45 (20-63) 9.7 ± 1.1 8.6 ± 1.4 45 (29-46) NA NA NA NA
Male/female sex, n 15/13 17/8 28/22 16/17 19/13 14/1 11/4 NA NA NA NA NA
Diagnosis HM HM HM HM HM + others AML/ALL AML/ALL HM NA NA NA NA
Myeloablative conditioning 0 Modified Bu/Cy Modified Bu/Cy + ATG 33 26 0 0 Cy + TBI + Flu NA NA NA NA
Reduced-intensity conditioning Flu + Mel + 0 0 0 6 15 15 0 NA NA NA NA
OKT3 + Thi
GVHD prophylaxis MMF MTX + CSA + MMF MTX + CSA + MMF ATG Cy + Tac + CSA CSA Cy + CSA + MMF ATG CSA + MMF CSA + CSA + MTX
MMF Cy + MMF
Allografts CD3/CD19- G-BM + G-PB G-BM + G-PB SS-BM SS-BM CD3/CD19-depleted SS-BM SS-BM SS-BM SS-BM SS-BM
depleted G-PB G-PB
Immune recovery, cells/L
CD3+ T cells
Day 30 post-SCT 8 at day +40 348 135 NA NA 870 226 31 146 30 195 477
Day 90 post-SCT 205 at day +100 1048 883 NA NA 983 652 413 at day +60 404 57 182 565
Day 180 post-SCT 369 at day +400 1037 1163 NA NA NA NA 691 470 196 499 700
CD3+CD4+ T cells
Day 30 post-SCT 5 at day +40 141 21 1 26 141 138 11 36 7 45 160
Day 90 post-SCT 70 at day +100 220 152 7 127 305 198 154 at day +60 86 36 127 170
Day 180 post-SCT 177 at day +400 276 163 69 194 NA NA 193 111 106 211 198
CD3+CD8+ T cells
Day 30 post-SCT 2 at day +40 115 98 1 22 730 85 11 102 42 73 280
Day 90 post-SCT 66 at day +100 645 672 17 181 678 447 193 at day +60 278 16 424 389
Day 180 post-SCT 193 at day +400 580 918 54 167 NA NA 409 413 51 408 500
CD3+CD4+ naïve cells
Day 30 post-SCT NA 90 6 0.7 3.45 3.9 5.3 0.45 NA NA NA NA
Day 90 post-SCT 28 at day +100 152 33 0.55 7.3 3.3 3.3 9 at day +60 NA NA NA NA
Day 180 post-SCT 115 at day +400 196 69 4.8 6.72 NA NA 24 NA NA NA NA
CD3+CD4+ memory cells
Day 30 post-SCT NA 131 20 2.6 24 136 134 4 NA NA NA NA
Day 90 post-SCT 79 at day +100 155 108 16 97 299 181 65 at day +60 NA NA NA NA
Day 180 post-SCT 205 at day +400 261 171 111 179 NA NA 48 NA NA NA NA
B cells
Day 30 post-SCT NA 13 13 5 0.95 39 24.6 0.5 NA NA NA NA
Day 90 post-SCT 100 at day +150 33 23 55 64 147 43 119 at day +60 NA NA NA NA
Day 180 post-SCT 275 at day +400 60 54 58 95 NA NA 104 NA NA NA NA
NK cells
Day 30 post-SCT 248 at day +20 NA NA 225 84 291 254 59 NA NA NA NA
Day 90 post-SCT NA NA NA 357 183 299 253 189 at day +60 NA NA NA NA
Day 180 post-SCT NA NA NA 290 172 NA NA 168 NA NA NA NA
HBMT indicates haploidentical blood and BM transplantation; HM, hematologic malignancy; AML, acute myelogenous leukemia; ALL, acute lympoblastic leukemia; ALT, alternative donor (1 antigen-mismatched family or unrelated donors) transplantation; CBIBT, unrelated cord blood grafted intrabone transplantation; NA, not available; Cy, cyclophosphamide; TBI, total body irradiation; Flu, fludarubine; Bu, busulfan; ATG, anti-human thymocyte immunoglobulin; Mel, melphalan; Thi, thiotepa; MMF, mycophenolate mofetil; CSA, cyclosporine A; MTX, methotrexate; Tac, tacrolimus; G-BM, G-CSF—primed BM; G-PB, G-CSF—mobilized peripheral blood stem cell grafts; SS-BM, steady-state BM. * Data coming from meeting abstract.
Effect of Recovered Immune Subsets on Outcomes after Haploidentical Transplantation
Disease (No. of Patients) [Reference]
Allografts
Conditioning Regimen
Reconstituted Immune Subsets
Potential Effects on Clinical Outcomes
Hematologic malignancies (27); other diseases (6) [23]
Hematologic malignancies (30); other diseases (2) [90]
Hematologic
malignancies (43) [24]
Selected CD34+ cells
Unmanipulated BM (13) and PBSCs (6); selected CD34+ cells (13)
G-BM + G-PB
Hematologic
malignancies (206) [18]
G-BM + G-PB
Hematologic malignancies (60) [59] G-BM + G-PB Hematologic malignancies (78) [6o] G-BM + G-PB Hematologic malignancies (21) [43] SS-BM
Hematologic
malignancies (22) [33]
Hematologic malignancies (98); nonmalignancies (33) [61]
Acute leukemia (30) [41]
Hematologic
malignancies (89) [91] Hematologic malignancies (47) [63]
G-PB + TCD
BM (78); G-PB (42); umbilical cord blood (11)
G-PB with CD3/CD19 depletion
MA (33) MA (32)
MA (43)
MA (206)
MA (60) MA (78) MA (21)
MA (22) MA (131) MA (30)
TCD and TCR allografts MA (89)
Thymic-dependent T cells: sjTREC <0.1/150,000T TREC <0.001/150,000T CD3+CD8+ T cells
CD56bright NK cells (9.27) and T:NK ratio at day 14 post-transplantation
Early recovered lymphocytes
Early recovered lymphocytes Early recovered lymphocytes Early recovered lymphocytes
CMV-specific CD8+ T cells (3 cyt+ cells/mL); CMV-specific CD4+ T cells (1 cyt+ cells/mL) Lymphoid DC2- cells
Low thymic function is associated with leukemia relapse.
Univariate analysis demonstrated superior survival in patients with CD8+CD3+ absolute counts above the 5th percentile of age-matched normal levels during the first year posttransplantation.
The patients with more CD56bright NK cells in the recovery stage had a higher survival rate (hazard ratio [HR], 0.406; P = .017) and the patients with a T/NK ratio >1.0 had a greater likelihood of getting aGVHD (HR, 3.436; P = .059) and chronic GVHD (HR, 3.925; P = .028). Multivariate analysis showed that patients with higher day 30 ALC (>300/mL) was associated with low relapse rate, low TRM, and superior survival in both adult and pediatric patients.
Unmanipulated PBSC (27); BM (20)
Nonmyleoablative (30); MA (17)
CMV-specific IFN ELISPOT (1000 spots/mL) Treg:CD4+ T cell ratio at day 14 post-transplantation
Patients with a day 30 ALC >200 cells/ pL had a markedly improved overall survival and event-free survival. The frequency of iNKT cells was significantly correlated with remission after transplantation. No CMV DNAnemia was evident.
A significant correlation was seen between the number of lymphoid DC2- cells on day +60 with patient survival.
Low TRM and virus reactivation were noted.
Multivariate analysis showed that a cutoff ratio value of 9% yielded the most accurate predictions of future aGVHD incidence. Treg:CD4+ T cell ratios of <9% predicted a significantly higher incidence of aGVHD compared with ratios >9% (P = .0082).
PBSCs indicate peripheral blood stem cells.
and leukemia relapse [2,3,71,87,88]. These results underscore the critical importance of improving immune recovery in both TCD and T cell—replete haploidentical HSCT settings.
ASSOCIATION OF RECOVERED IMMUNE CELLS WITH TRANSPLANTATION OUTCOMES
A number of studies have evaluated the association of immune recovery with clinical outcomes after haploidentical transplantation (Table 2) [18,24,59,60]. A higher absolute lymphocyte count at day 30 post-transplantation (ALC-30; >300 cells/pL) is associated with lower incidences of relapse and transplantation-related mortality (TRM) and superior leukemia-free survival and overall survival in both adult and pediatric patients after unmanipulated haploidentical blood and BM transplantation [18,59,60]. Using the unmanipulated haploidentical HSCT protocol, the Peking University group demonstrated a survival advantage for patients with high early (day +14) levels of CD56bright NK cells after unmanipulated haploidentical HSCT [24]. In Spain, Martin et al. [89]
confirmed this result using unmanipulated haploidentical BM grafts after RIC. Other factors, including lower numbers of recovered CD3+CD8+ T cells and lymphoid mDC2 cells, may contribute to inferior survival [41-90]. Fujiokaet al. [63] reported that a Treg:CD4+ T cell ratio <9% is predictive a significantly higher incidence of acute GVHD compared with a ratio >9% (P = .0082) after unmanipulated blood or BM transplantation. This finding awaits confirmation in other haploidentical HSCT protocols, such as transplantation with CD3/CD19-depleted allografts [8] and BM transplantation with high-dose post-transplantation cyclophos-phamide [71].
Another study investigated multiple parameters of T cell immune reconstitution in 89 patients who underwent hap-loidentical HSCT. Using a receiver operating characteristic curve analysis of cytomegalovirus (CMV)-specific IFN enzyme-linked immunosorbent spot (ELISPOT) assay results at day 30 to 90, Noviello et al. [91] found that cutoff values of 1000 spots/mL allowed researchers to identify those patients
who did not reactivate the virus at later time points with high specificity (>95%). Strikingly, the 2-year TRM rate was 32% in patients with an ELISPOT finding of <1000 spots/mL and 0% in those with >1000 spots/mL (P < .05). This interesting finding warrants the use of a predictive biomarker in larger multicenter series to investigate whether a CMV-specific IFN- ELISPOT cutoff value of 1000 spots/mL is a strong surrogate biomarker for TRM. When Clave et al. [23] evaluated thymic function in a group of pediatric recipients, they found that 6 months after CD34-selected HLA-haploidentical HSCT, a TREC value below the limit of detection (<0.1 per 150,000 CD3+ T cells for sjTREC and <0.001 per 150,000 CD3+ T cells for TREC) was associated with an increased risk of relapse [23].
In summary, prospective studies with larger patient populations are needed to draw definitive conclusions regarding the impact of recovered immune cells on clinical outcomes in T cell—replete and TCD nonmyeloablative or myeloablative haploidentical settings [3,8,92]. These studies are important to the prediction of such clinical outcomes as infection rates, relapse, and TRM [37,62]. Shimoni [62] suggested that the ALC-30 has long been a simple test of immune recovery that can predict outcome after autologous HSCT as well as allogeneic and haploidentical HSCT [18,59,60]. It is easy, reproducible, and requires no special expertise. Thus, the measurement of ALC-30 may have practical significance in applying interventions to facilitate immune reconstitution as described previously for hap-loidentical transplants [18,42,59,60].
COMPARISION OF IMMUNE RECOVERY BETWEEN HAPLODENTICAL AND OTHER TRANSPLANTATION MODALITIES OR GRAFT OPTIONS
Several published prospective and retrospective studies have highlighted the differences in immune reconstitution after unmanipulated haploidentical HSCT versus HLA-matched sibling donor transplantation (MSDT), matched unrelated donor transplantation (MUDT), or umbilical cord blood transplantation (Table 1). Chang et al. [25] found that compared with HLA-matched recipients, haploidentical recipients had lower counts of T cells (particularly CD4 T cells) and DC subsets before day +90, as well as greater CD28 expression on CD4+ and CD8+ T cells, whereas T cells were equally functional in the 2 patient groups at day +30. In addition, Dominietto et al. [93] recently demonstrated early delayed immune reconstitution after haploidentical transplantation (HIT) compared with MSDT [93]. Several lines of evidence suggest an association between improved immune recovery and better transplantation outcomes [13,24,94-96]; however, in our center, transplantation outcomes regarding overall survival and leukemia-free survival were comparable after HIT and MSDT, although unmanipulated HIT was characterized by delayed early reconstitution of T cells and DCs, as well as a higher incidence of CMV antigenemia [86,87]. Several factors may account for this finding. Compensatory expansion of monocytes, NK cells, and cytotoxic T lymphocytes (CTLs) [25], especially CMV-pp65 peptide-specific CTLs with the central memory CD45RO+CD62L+ cell phenotype, accompanies the recovery of CD8+ T cells. This cell population may proliferate and differentiate into effector memory T cells stimulated with CMV antigen and may contribute to a reduced incidence of CMV disease [31,47]. Other possible contributing factors include the preemptive management of CMV antigenemia, a superior graft-versus-leukemia effect associated with transplantation of haploidentical versus
HLA-identical sibling donor grafts for high-risk acute leukemia [97], and improvements in minimal residual disease-directed relapse prophylaxis using modified donor lymphocyte infusion [66,98-100].
In a detailed prospective analysis of immune reconstitution in MUDT and HIT with CD3/CD19-depleted grafts in 30 pediatric patients, Pérez-Martínez et al. [41] found more rapid CD8 and CD8 memory T cell recovery during the first month in HIT compared with MUDT (730 i 208 versus 85 i 37 cells/mL and 723 i 274 versus 83 i 29 cells/mL, respectively; P < .05). CD80 expression in the DC1-DC2-subset was higher in HIT patients than the CD80 expression in the DC1-DC2- subset in MUDT patients during the first
2 months after HSCT (respectively, 26 i 6.98 vs 5 i 1.4/mL, P = .04 and 28 i 12 vs 2 i 0.8/mL, P = .05); however, the reconstitution of NKT cells, NK cells, and B cells was comparable in the 2 groups [41]. Szabolcs et al. [101] reported an association between CD8+ lymphocytosis and effector memory phenotype and IFN secretion in children who developed de novo opportunistic infections. Brown et al. [102] presented data on recent thymic emigrants and CMV-specific immune responses demonstrating associations between higher TREC and CMV-specific immune responses and improved survival. Sauter et al. [103] found that an ATG-free regimen after double cord blood transplantation could result in more rapid CD4+ T lymphocyte reconstitution, and reported no infection-related deaths after day +120, suggesting a correlation between immune recovery and infection-related death. Continuing new insights into the mechanisms underlying differences in the kinetics of immune reconstitution after HIT compared with MSDT and MUDT may help guide the design of useful strategies to accelerate immune recovery in HIT settings.
STRATEGIES TO IMPROVE IMMUNE RECONSTITUTION AFTER HAPLOIDENTICAL HSCT
Many of the currently available approaches to accelerating immune recovery, including IL-7, keratinocyte growth factor, growth hormone, and adoptive transfer of immune cell subsets, have been reviewed elsewhere [12,19,37,66,104-106]. Here we discuss only recent informative updated trials and reports (Table 3); data reported between 2012 and 2013) regarding the promotion of immune reconstitution, particularly in haploidentical transplantation settings [53,65,69,107].
Cytokines
Among the cytokines used to enhance T cell reconstitution, IL-7 certainly tops the list. In a phase 1 trial, Perales et al. [65] treated 12 patients who underwent MSDT or MUDT with escalating weekly doses of IL-7 for a 3-week period. Three patients received 10 mg/kg, 6 patients received 20 mg/kg, and
3 patients received 30 mg/kg. IL-7 was well tolerated overall, with only 1 patient developing acute skin GVHD. Compared with baseline values, levels of recovered CD3+, CD4+, and CD8+ Tcells increased by 4.3-, 6.1-, and 4.3-fold, respectively. IL-7 also enhanced functional T cell responses and TCR diversity. Unfortunately, IL-7 appeared to have no significant effect on CD4+CD25+FoxP3+ T cells, NK cells, or B cells, although the data that IL-7 significantly enhances T cell recovery are exciting [65]. Based on the foregoing findings, other approaches applied alone or in combination with IL-7 to promote the reconstitution of all immune subsets merit investigation in both HLA-matched and haploidentical transplantation protocols [2,3,87,88].
Recent Informative* Trials and Reports Regarding Strategies for Enhancing Immune Recovery
Strategy
Transplantation Protocol
Key Points
Reference
Approaches used in preclinical model IL-15
Depletion of naive lymphocyte with Fas ligand
Approaches used in clinical trial or report IL-7
HIT mice model
HIT mice model
Escalating-dose DLI
Modified DLI
Preemptive immunotherapy with NK cells
Adoptive transfer of suicide gene-tranduced donor T cells
Adoptive transfer of donor-derived CMV-specific T cells
Adoptive transfer of trivirus-directed T cells
IL-15 can be used for enhancing antileukemia effect of haploidentical HSCT, which requires presence of donor-derived T cells, especially CD8+ T cells. The proposed approach eliminates severe GVHD in T cell —replete transplants, preserves the tumor lytic activity of mismatched donor T cells, maintains T cell reactivity to third-party antigens, and, facilitates HSC engraftment.
MRDT and MUDT Treatment 12 patients with IL-7 increases T cell recovery, induces T cell responses, and enhances TCR diversity. IL-7 have no significant effect on Treg, NK or B cells.
HIT This study suggests that DLI, using an excalating-dose
regimen, can be safely given to mismatched transplant recipients with a high rate of molecular remission and a low risk of GVHD.
HIT and MRDT Eight hundred and fourteen patients were enrolled, this
study suggest that risk stratification-directed modified DLI may reduce relapse and improve survival of subjects with standard-risk acute leukemia after HSCT, although the effect of DLI on immune reconstitution need to be investigated.
HIT In this prospective phase II study, the results suggest that
NK cell activation/expansion may be required to attain clinical benefit, while careful consideration must be given to the number of T cells infused.
HIT Thymopoiesis is reactivated after the infusion of gene-
modified lymphocytes, enhancing the generation of a protective T cell compartment that is able to reduce the incidence of infectious and control of GVHD.
HIT, MSDT, and MUDT Infusion of CMV-specific T cell early posttransplant reduced the need for pharmacotherapy without increased rates of CMV-related organ damage and increasing acute and chronic GVHD.
HIT, MSDT, and MUDT In ten patients, trivirus-directed T cells administration
produced completed virological responses in 80% cases and a decrease in viral load was associated with an increase in the frequency of T cell directed against the infecting virus. No toxicities were observed.
Sauter et al. 2013 [58] Askenasy et al. 2013 [49]
Perales et al. 2012 [65] Inneset al. 2013 [55]
Yan et al. 2012 [98]
Stern et al. 2013 [108] Vaglo et al. 2012 [65] Blythet al. 2012 [69] Gerdemman et al. 2013 [63]
MRDT indicates HLA-matched related donor transplantation; DLI, donor lymphocyte infusion.
* Published between 2012 and 2013.
Using a clinically relevant haploidentical murine transplantation model, Sauter et al. [58] found that IL-15 administration significantly increased CD8+ T cell and NK cell reconstitution and also improved graft-versus-leuke-mia/graft-versus-tumor activity in recipients of hap-loidentical HSCT. These authors also demonstrated that IL-15 increased intracellular IFN secretion in undivided and slowly proliferating CD8+ T cells in recipients of car-boxyfluoresceinsuccinimidyl ester—labeled T cells, with no change in the IFN-secreting alloreactive/rapidly prolifera-tive cell population [58].
The efficiency, safety, and feasibility of using IL-15—stimulated CD3/CD19-depleted stem cells or IL-15—activated cytokine-induced killer cells to treat relapse after haploidentical transplantation have been investigated, albeit only in small patient populations [51,57]. Pfeiffer et al. [57] reported that in 8 patients, recovered CD56+ CD3+/CD4+ and CD3+/CD8+ lymphocyte counts at 15 to 30 days after receipt of IL-15—stimulated stem cell boosts were similar to or even higher than those before the initiation of chemotherapy.
Preemptive Transfer of NK Cells
Adoptive immunotherapy with allogeneic purified NK cell products might exert graft-versus-tumor alloreactivity with little risk of GVHD. In a prospective phase 2 study, Stern et al.
[108] administered purified NK cell products to high-risk patients treated with haploidentical TCD HSCT. Sixteen patients received a total of 29 NK cell infusions on days +3, +40, and +100 post-transplantation. The median dose of infused NK cells per product was 1.21 x 107/kg (range, 0.3-3.8 x 107/kg). Four of the 16 patients were alive at the time of this report, at a median follow-up of 5.8 years. Rapid reconstitution of lymphocyte subsets at day +100 was observed, with a median CD8 T cell count of 266 cells/mL and a median CD4 T cell count of 181 cells/mL. These promising results warrant further study to determine the optimal dose and timing of NK cell immunotherapy for antileukemia activity.
Genetically Modified Donor T Cell Infusion
Several researchers have investigated the use of suicide gene—transduced donor T cells to enhance immune reconstitution while controlling GVHD. In a multicenter phase 1/2 clinical trial, purified herpes simplex thymidine kinase suicide gene—transduced donor T cells (TKpos cells) were infused serially into 28 adult patients with a hematologic malignancy after CD34-selected haploidentical transplantation [70]. Twenty-two of the 28 patients who received the purified TKpos cells exhibited a rapid recovery of T cells associated with a concomitant improvement in clinical outcomes and reduced TRM. Patients who did not receive TKpos
Figure 1. A proposed prognostic immune subset—directed strategy to improve immune reconstitution after HIT. On day 0, in vitro alloreactive T cell-and/or B cell—depleted allografts, including NK cells, HSCs, Tregs, and myeloid-derived suppressor cells (MDSCs), were infused to the recipients who received a conditioning regimen to decrease the incidence of GVHD and enhance immune reconstitution. After transplantation, the kinetics of immune recovery were monitored. For patients with rapid immune recovery (as demonstrated by ALC-30 > 300/mL or another parameter predictive of rapid immune recovery), no interventions were required, and immune reconstitution was monitored only. For patients with delayed immune recovery (ALC-30 < 300/mL or other parameters predictive of delayed immune recovery), interventions, including IL-7 and/or adoptive transfer of expanded progenitor T cells or virus-specific donor T cells, were implemented to improve immune reconstitution. MHC, major histocompatibility complex.
cells failed to attain T cell immune reconstitution and experienced dismal outcomes, owing mainly to infectious complications. Impressively, further follow-up of these patients revealed that transfer of TKpos cells not only enhanced the regeneration of CD3+ T cells, but also increased counts of the CD4+ T cell subset and the T cell subset with a naive phenotype (CD62L+CD45RA+CD31+), as well as sjTREC counts, in parallel to the increase in the naive T cell subset. Strikingly, an increase in serum IL-7 level was observed early after the TKpos T cell add-back, followed by a concomitant rise in peripheral T cell counts. Computed tomography—based analysis revealed an increase in thymic tissue even in elderly patients. Additional experiments demonstrated that the antiviral activity of the newly generated T cells was fully maintained.
The biological mechanism responsible for these unexpected findings is unclear. The transient increase in IL-7 might be a contributing factor, as described by Perales et al. [65] in a phase 1 trial, but is likely insufficient to induce long-lasting thymopoiesis. The findings of Vago et al. [70] might suggest new approaches to accelerating immune reconstitution in patients not only after haploidentical transplantation of CD34+ stem cells, but also in other transplantation settings, and particularly in elderly patients [2,3,67,71,88].
Adoptive Transfer of Pathogen-Specific T Cells
Gerdemann et al. [49] recently developed a new rapid and simplified manufacturing strategy in which DCs nucleofected with DNA plasmids encoding a range of immunodominant and subdominant viral antigens from adenovirus (Adv), CMV, or Epstein-Barr virus (EBV) are used to generate trivirus-specific T cells. In a recent study, trivirus-directed
T cells were administered to recipients of haploidentical (n = 5), matched unrelated (n = 3), mismatched unrelated (n = 1), or matched related (n = 1) transplants with active CMV (n = 3), Adv (n = 1), EBV (n = 2), EBV + Adv (n = 2), or CMV + Adv (n = 2) infections. The cells produced complete virologic responses in 80% of recipients, including all patients with dual infections. In each case, a decrease in viral load was correlated with an increase in the frequency of T cells directed against the infecting virus(es); both immediate and delayed toxicities were absent [68]. The authors suggested that a DNA plasmid-based approach might increase both the feasibility and applicability of T cell therapy.
Little data are available on vaccine responses in patients undergoing HIT [109]. More recently, several other approaches, including the depletion of naive lymphocytes using Fas ligand [53] and the infusion of CD62L- memory Tcells [107], have been used in preclinical models to enhance immune reconstitution after transplantation. Additional studies are warranted to investigate whether these preclinical approaches can be translated into clinical trials.
FUTURE DIRECTIONS
Delayed immune reconstitution is an important issue in haploidentical HSCT. Substantial progress has been made in the field, including insight into recovered immune subsets that can predict outcomes and new strategies for accelerating immune recovery. These advances make it possible to establish a prognostic immune subset—directed strategy to improve immune recovery in haploidentical settings (Figure 1). To achieve this goal, prospective, multicenter trials are needed to investigate whether a low ALC-30 or other simple immune parameters can be used to identify which patients should be treated to improve immune recovery. In
addition, pilot studies should be conducted to evaluate the most effective immune reconstitution-accelerating strategy or combination of approaches. Although promising survival has been achieved with the establishment of many hap-loidentical transplantation protocols, we believe that a predictive immune subset-guided strategy to accelerate immune recovery might further improve transplantation outcomes in the future, making haploidentical transplantation a routine strategy for patients requiring HSCT.
ACKNOWLEDGMENTS
The authors would like to express their apologies to those whose important work was not cited in this review. They thank San Francisco Edit (www.sefedit.net) for their assistance in editing the manuscript.
Financial disclosure: Support was provided by the Key Program of the National Natural Science Foundation of China (Grant 81230013), the ChangJiang Scholars Program, and the Bejing Municipal Science and Technology Commission (Grants Z121107002612035 and Z121107002812033).
Conflict of interest statement: There are no conflicts of interest to report.
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