Long-term Immune Reconstitution Research Articles

CD4 nadir and neurocognitive trajectories in people living with HIV

AbstractHuman immunodeficiency virus-associated neurocognitive disorders persist in the combination antiretroviral therapy era. CD4 nadir is a well-established predictor of cognition cross-sectionally, but its impact on longitudinal neurocognitive (NC) trajectories is unclear. The few studies on this topic examined trajectories of global cognition, rather than specific NC domains. The current study examined CD4 nadir in relation to domain-specific NC decline. 132 HIV + adults from the Temple/Drexel Comprehensive NeuroHIV Center, Clinical and Translational Research Support Core Cohort were administered comprehensive NC assessments longitudinally, with last visit occurring an average of 12 years after CD4 nadir. Linear mixed models were used to examine CD4 nadir in relation to longitudinal NC trajectories in three empirically identified NC domains: speed/executive function (S/EF), visuospatial memory (VM), and verbal fluency (VF). CD4 nadir was associated with change in VF (p = 0.020), but not with S/EF or VM. Specifically, those with CD4 nadir < 200 demonstrated increasing VF over time (p = .002), whereas those with CD4 nadir > 200 demonstrated stable VF (p = .568), though these differing trajectories may partly reflect regression to the mean or differential practice effect. CD4 dynamics over time were analyzed as potential mechanisms for the identified associations, with mixed findings. While low CD4 nadir has been associated with weaker neurocognition among people living with HIV, the results of this study suggest that low CD4 nadir is not associated with ongoing decline a decade later. Nadir-related deficits in VF may be stable or even improve over time, possibly reflecting the beneficial cognitive effects of long-term treatment and immune reconstitution.

Long-Term Efficacy and Immune Reconstitution with Bendamustine As a Lymphodepleting Agent for Axicabtagene Ciloleucel (Axi-Cel) in Patients with Refractory or Relapsed Large B-Cell Lymphoma (LBCL)

Introduction: Lymphodepletion (LD) is crucial for the expansion and persistence of CAR T cells. Fludarabine in combination with cyclophosphamide (FC) is the LD standard regimen. A national fludarabine shortage in 2022 necessitated the exploration of alternative LD regimens. Bendamustine (benda) is a purine analog and an alkylating chemotherapy that has been used as an LD agent for tisagenlecleucel (Ghilardi et al.,2022). However, there is no data for benda LD in other commonly used CART products for relapsed or refractory non-hodgkin lymphoma (rel/ref NHL). Early safety, efficacy, and expansion kinetics data were presented previously (Bharadwaj, ASH 2022). We are now reporting long-term efficacy and immune reconstitution data. Methods: 84 consecutive patients with relapsed/refractory LBCL treated with axi-cel were included in this single-institution study. 27 patients who received Bendamustine lymphodepletion (90mg/m2 on days -4 and -3) were compared to a preceding 57-patient FC cohort. Response, survival, and cytopenia data were collected closest to the 12-month time point for all patients who had not progressed. Response was assessed using the Lugano 2014 criteria, while toxicities were evaluated using the CTCAE version 5.0 and the ASTCT grading. To reduce the influence of confounding variables in this study, a covariate balance propensity score weighting approach (IPTW) was implemented. Covariates included age, sex, presence of bridging therapy, IPI score at apheresis, number of prior therapy lines, absolute lymphocyte count at leukapheresis, prior auto SCT, and pre-lymphodepletion LDH, ALC, ANC, and platelet count. Continuous outcomes were compared using the Mann-Whitney U test, categorical outcomes using chi-square tests, and time-to-event outcomes using a log-rank test weighted in the IPTW sample. Statistical significance was considered when p < 0.05. All analyses were conducted in R 4.3.0. Results: There was no significant difference in the best complete response (CR) rate and best overall response rate (ORR) between the two groups, with a CR rate of 71.9% for FC and 74% for bendamustine (p = 0.182), and an ORR of 89.4% for FC and 85.1% for bendamustine (p = 0.502). For the FC group, 9-month progression-free survival (PFS) and overall survival (OS) were 63.16% (95% CI: 49.29% to 74.19%) and 78.95% (95% CI: 65.92% to 87.45%), respectively with a median follow-up of 15.5 months (IQR: 13.6, 16.8). 9-month PFS and OS point-estimates for the bendamustine group were 70.37% (95% CI: 49.4% to 83.94%) and 77.41% (95% CI: 56.41% to 89.18%), respectively, with a median follow-up of 9.5 months (IQR: 8.6, 9.8). No significant difference was found between groups in either 9 months PFS ( P=0.055) or 9 months OS ( P=0.6290). Median PFS and OS have not been achieved for either group [Figure 1]. Bendamustine was effective in achieving severe lymphodepletion, with a median absolute lymphocyte count (ALC) nadir of 0.11 K/ul (IQR: 0.04, 0.17) occurring within one day after axi-cel infusion. In comparison, the FC group had a lower median ALC nadir of 0.03 K/ul (IQR: 0.01, 0.06) occurring on the day of axi-cel infusion ( P=0.01). Both cohorts showed a similar pattern of lymphocyte recovery over 1, 3, and 6 months. Median ALC counts at 9-12 months post-infusion indicated better lymphocyte recovery in the bendamustine group with a median ALC of 1.55 K/ul (IQR: 1.04, 1.94) compared to 0.96 K/ul (IQR: 0.58, 1.23) in the FC group ( P=0.043) [Figure 2]. There was no significant difference in the recovery of absolute neutrophil count, hemoglobin, and platelets. Updated efficacy analysis and immune reconstitution data including T-cell, B-cell, and NK cell recovery will be reported at the time meeting, as all patients will have completed at least one year of follow-up by September 2023. Discussion: Bendamustine lymphodepletion prior to axi-cel therapy showed comparable efficacy and safety outcomes to the standard FC regimen in relapsed/refractory LBCL patients. Bendamustine demonstrated effective lymphodepletion and better lymphocyte recovery at 9-12 months post-infusion compared to FC cohort. These findings suggest that bendamustine may be an acceptable alternative lymphodepleting regimen. Prospective studies, ideally randomized control trial, are warranted.

Long-Term Analysis of Anti-CD20mab + HDMP for the Treatment of Patients with Chronic Lymphocytic Leukemia

Background: The addition of an anti-CD20 monoclonal antibody (mAB) to high dose methylprednisolone (HDMP) has been demonstrated to be an effective, non-myelosuppressive treatment (Tx) regimen for patients (Pts) with chronic lymphocytic leukemia (CLL). (Castro et al., 2009) We performed a single institution retrospective analysis of Pts treated with an anti-CD20 mAB and HDMP evaluating long-term efficacy, safety, and immune reconstitution. Methods: 102 Pts treated between the years of 2001 and 2015 were evaluated. Tx consisted of HDMP 1gm/m2 IV on days 1, 2, and 3 of each cycle. Rituximab (R) was administered as either 375 mg/m2 weekly; 750 mg/m2 daily on days 1, 2 and 3; or 750 mg/m2 weekly times during cycle one and daily times three for cycle two and three. Obinutuzumab (G) and ofatumumab (O) were administered per FDA standard dosing. Pts received 3-6 cycles of Tx. Some Pts who achieved a partial response (PR) to initial therapy underwent alemtuzumab (Alem) consolidation, 10mg, administered three times a week for a total of eight weeks. All Pts received anti-microbial prophylaxis consisting of fluconazole, bactrim and acyclovir. Response assessment was performed using iwCLL criteria. Results: The Pts had a median age of 66 (range; 40-85). Rai stage at time of Tx initiation: I 6%; II 43%; III 17%; Stage IV 34%. 63% of Pts were previously treated. The median number of previous Txs was 2 (range; 1-5). 55% of Pts had been treated with immunotherapy alone, 67% with chemoimmunotherapy, and 13% of Pts with a BCL2 inhibitor. Pts harbored the following cytogenetic abnormalities: del(13q) 46%; trisomy 12 30%; del(11q) 22%; normal 13%; del(11q) 22%; del(17p) 19%; and complex in 26%. 58% of Pts harbored an unmutated immunoglobulin heavy chain (IGHV). 84% of Pts were treated with R, 11% with G, and 5% with O. 96 Pts (94%) had a response to Tx (OR). 18 Pts (18%) achieved a complete response (CR); 78 Pts (76%) achieved a PR; and six Pts (6%) had stable disease (SD). Seventeen Pts underwent Alem consolidation. Twelve Pts (71%) achieved a CR; four Pts (24%) achieved a PR; and one patient (6%) had SD after Alem consolidation. With a median follow-up of 114 months, the median progression free survival (PFS) for the entire cohort was 27.1 months. The median time to next treatment (TTNT) for the entire cohort was 30.0 months. For Pts who received Alem consolidation, the median PFS was 90.1 months, and the median TTNT was 97.8 months. (Figure 1A) Employing cox proportional hazards model, factors associated with improved PFS were IGHV mutated (HR 0.38; 95% CI 0.18 to 0.79); CR (HR 0.17; 95% CI 0.05 to 0.47); and Alem consolidation (HR 0.21; 95% CI 0.09 to 0.46). Factors associated with improved TTNT was presence of del13q (HR 0.41; 95% CI 0.18 to 0.93); IGHV mutated (HR 0.36; 95% CI 0.13 to 0.92); CR (HR 0.12; 95% CI 0.03 to 0.37); and Alem consolidation (HR 0.24; 95% CI 0.10 to 0.53). 9% Pts developed infection requiring hospitalization, and 5% Pts developed an opportunistic infection. Immediately after Tx, serum IgA, IgG and IgM levels decreased (mean IgA pre-Tx 69.4mg/dL vs post-Tx 43.0mg/dL; p <0.0001; mean IgG pre-Tx 738.0mg/dL vs post-Tx 494.3mg/dL; p <0.001; mean IgM pre-Tx 94.8mg/dL vs post-Tx 44.5mg/dL; p = 0.07). At approximately 36- and 48-months serum IgA and IgG levels returned to normal levels, respectively. (Figure 1B) 57% of Pts received supplemental intravenous immunoglobulin, thus we investigated factors associated with recovery of serum IgA levels at 36 months. We found that only disease status at 36 months impacted serum IgA levels. (Mean IgA without progression at 36 months 74.7mg/dL vs 44.9 mg/dL with progression at 36 months; p = 0.035). Conclusions: In our analysis of Pts with CLL treated with an anti-CD20 mAB + HDMP we found a high OR of 94% and median PFS of 27.1 months. This compares favorably to traditional low intensity chemoimmunotherapy approaches for CLL. Pts who received Alem consolidation had durable remissions, with a median PFS of 90.1 months. The overall incidence of infectious complications was manageable, with normalization of serum IgA and IgG three and four years after completion of therapy. Patients without progression at 36 months were found to have higher serum IgA levels. While the Tx landscape for CLL has dramatically changed, the addition of HDMP to an anti-CD20 mAB can be a Tx option for patients desiring short course of Tx, requiring bridging to more definite therapy, or debulking strategy prior to venetoclax based Tx. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Addition of a Single Low Dose of Anti T-Lymphocyte Globulin to Post-Transplant Cyclophosphamide after Allogeneic Hematopoietic Stem Cell Transplant: A Pilot Study.

Correlation between risk of graft-versus-host disease (GvHD) and CD3+ counts within the peripheral blood stem cell graft has recently been reported in the setting of post-transplant cyclophosphamide (PT-Cy). We aimed to investigate the benefit of the addition of a single dose of anti-T lymphocyte globulin (ATLG 5 mg/kg) to PT-Cy in this setting. Starting in 2019, all patients receiving PBSC transplant containing CD3+ counts above 300 × 106/kg (study group) received a post-transplant dose of ATLG in addition to standard PT-Cy. The study was designed as a real-life analysis and included all consecutive Hematopoietic Stem Cell Transplantation (HSCT) recipients according to the above-mentioned inclusion criterion (n = 21), excluding cord blood and bone marrow donors. Using a 1:2 matched-pair analysis, we compared the outcomes with a historical population who received PT-Cy only (control group). We found a delayed platelet engraftment (29% vs. 45% at 30 days, p = 0.03) and a non-significant trend toward higher risk of poor graft function (29% vs. 19%, p = 0.52). The addition of ATLG impacted long-term immune reconstitution on the CD4+ subsets, but this did not translate into higher rate of relapse or viral infection. Acute GvHD was not significantly impacted, but 1-year cumulative incidence of chronic GvHD was significantly lower in the study group (15% vs. 41%, p = 0.04). Survival outcomes were comparable. In conclusion PT-Cy and ATLG was overall safe and translated into a low rate of chronic GvHD incidence.

Role of Brain Arterial Remodeling in HIV-Associated Cerebrovascular Outcomes.

As the life expectancy of people living with HIV (PLWH) on combination antiretroviral therapy (cART) increases, so does morbidity from cerebrovascular disease and neurocognitive disorders. Brain arterial remodeling stands out as a novel investigational target to understand the role of HIV in cerebrovascular and neurocognitive outcomes. We therefore conducted a review of publications in PubMed, EMBASE, Web of Science and Wiley Online Library, from inception to April 2021. We included search terms such as HIV, cART, brain, neuroimmunity, arterial remodeling, cerebrovascular disease, and neurocognitive disorders. The literature shows that, in the post-cART era, PLWH continue to experience an increased risk of stroke and neurocognitive disorders (albeit milder forms) compared to uninfected populations. PLWH who are immunosuppressed have a higher proportion of hemorrhagic strokes and strokes caused by opportunistic infection and HIV vasculopathy, while PLWH on long-term cART have higher rates of ischemic strokes, compared to HIV-seronegative controls. Brain large artery atherosclerosis in PLWH is associated with lower CD4 nadir and higher CD4 count during the stroke event. HIV vasculopathy, a form of non-atherosclerotic outward remodeling, on the other hand, is associated with protracted immunosuppression. HIV vasculopathy was also linked to a thinner media layer and increased adventitial macrophages, suggestive of non-atherosclerotic degeneration of the brain arterial wall in the setting of chronic central nervous system inflammation. Cerebrovascular architecture seems to be differentially affected by HIV infection in successfully treated versus immunosuppressed PLWH. Brain large artery atherosclerosis is prevalent even with long-term immune reconstitution post-cART. HIV-associated changes in brain arterial walls may also relate to higher rates of HIV-associated neurocognitive disorders, although milder forms are more prevalent in the post-cART era. The underlying mechanisms of HIV-associated pathological arterial remodeling remain poorly understood, but a role has been proposed for chronic HIV-associated inflammation with increased burden on the vasculature. Neuroimaging may come to play a role in assessing brain arterial remodeling and stratifying cerebrovascular risk, but the data remains inconclusive. An improved understanding of the different phenotypes of brain arterial remodeling associated with HIV may reveal opportunities to reduce rates of cerebrovascular disease in the aging population of PLWH on cART.

Long-Term Immune Reconstitution and Outcome After HLA-Nonidentical T-Cell–Depleted Bone Marrow Transplantation for Severe Combined Immunodeficiency: A European Retrospective Study of 116 Patients

AbstractWe have performed a retrospective analysis of the development of T- and B-cell functions after HLA-nonidentical T-cell–depleted bone marrow transplantation (BMT) performed in 193 patients with severe combined immunodeficiency (SCID) at 18 European centers between December 1982 and December 31, 1993. One hundred sixteen of 193 patients were alive with evidence of engraftment 6 months after BMT. Development of T-cell function occurred earlier than B-cell function and was achieved more frequently up to the time of last follow-up. The median time to achieve normal T-cell function was 8.7 months, whereas the median time to achieve normal B-cell function was 14.9 months. Twenty-four patients died later than 6 months post-BMT, mainly due to chronic graft-versus-host disease (cGVHD) and/or viral infection. Absence of T-cell reconstitution 6 months after BMT, unlike absence of B-cell reconstitution, was associated with a poor outcome. Two additional factors were associated with a poor outcome: presence of cGVHD 6 months after BMT and B− SCID versus B+ SCID. However, two of these three factors remained as significant prognostic factors in a multivariate analysis: the absence of T-cell function and the presence of cGVHD 6 months after BMT. Analysis of the factors influencing the development of immune reconstitution showed that T- and B-cell functions occurred earlier and more frequently in B+ SCID versus B− SCID patients. Acute GVHD was associated with a slower development of T-cell function at 6 months, and cGVHD had a negative influence on the development of T-cell function afterwards, but neither acute nor chronic GVHD was found to influence the development of B-cell function. Once engraftment occurred, whether patients had or had not received Busulfan in the conditioning regimen did not influence the kinetics and quality of T-cell function development. In a multivariate study, two factors were found to influence the T-cell function 6 months after BMT: type of SCID and acute GVHD. The results of this retrospective analysis should lead to new protocols adapted to SCID disease, considering that disease-related as well as BMT-related parameters influence the development of immune function and thereby long-term outcome after HLA-nonidentical T-cell–depleted BMT.

Allogeneic Hematopoietic Stem-Cell Transplantation in Patients with GATA 2 Deficiency: Influence of Donor Stem Cell Source and Post-Transplantation Cyclophosphamide

Background: We recently reported on the single-institution experience with allogeneic hematopoietic stem cell transplantation (HSCT) in 22 consecutive patients with GATA2 deficiency or the MonoMAC syndrome and observed a disease-free survival of nearly 90%. However, despite 10/10 HLA match of matched related donors (MRD) and matched unrelated donors (URD), there was a 25% incidence of grades III-IV acute graft-versus-host disease (GVHD) with tacrolimus/methotrexate (Tacro/MTX). In contrast, there was no grade III-IV acute GVHD in the haploidentical related donor (HRD) recipients, all of whom received GVHD prophylaxis with post-transplantation cyclophosphamide (PT/Cy) followed by tacrolimus/mycophenolate (tacro/MMF). Based on this initial experience, the protocol was subsequently amended to incorporate PT/Cy in all patients. We now report on this expanded cohort of a total of 59 patients, representing the largest experience with HSCT for GATA2 deficiency. Methods: This single-institution study was conducted at the National Institutes of Health Clinical Center between 2013 and 2020 (ClinicalTrials.gov Identifier: NCT01861106). Patients between 12 to 60 years of age were eligible if they had a deleterious mutation in the GATA2 gene, or clinical picture of the MonoMAC syndrome. Although the primary endpoints were engraftment and reversal of the clinical phenotype, a secondary endpoint was added to determine if PT/Cy in the MRD and URD reduced the incidence of grade III-IV aGVHD without compromising the overall and disease-free survival. Immune reconstitution at 100 days, 6, 12, 24, 36 months was analyzed. Results: 59 patients (median age at diagnosis 24 years; IQR 20-32) with GATA2 deficiency or the MonoMAC syndrome underwent allogeneic HSCT. MRD and URD recipients received busulfan for four days (targeted to an AUC 3600-4800) and fludarabine, whereas HRD recipients received two days of low dose cyclophosphamide, 5 days fludarabine, 200cGY total body irradiation, and two or three days of busulfan depending upon the presence or absence of clonal cytogenetic abnormalities. Donor sources included: 11 MRD, 31 URD, and 17 HRD. Median follow-up was 2 years [IQR 1-4], 88% (52/59) are currently alive. Seven deaths have occurred, including: persistent AML (n=1); infection (n=4); poor graft function and cardiac arrest (n=1) and from HPV-associated metastatic cancer (n=1). All of the patients who received HRD transplant are alive. Median time to neutrophil engraftment was 15 days (IQR 13-17) and 19.5 days (IQR 16-25) for platelets. Ninety three percent (55/59) of patients had blood test results after 100 days post-transplant to assess immune reconstitution; eighty one percent (45/55) had normal absolute monocyte counts, 67% (37/55) had normal absolute NK cell counts and 89% (49/55) had normal B cells counts. There was one case of primary graft failure in a recipient of an URD-transplant, and one secondary graft rejection in a recipient of HRD-transplant. Fifty-four percent (32/59) of patients had pre-HSCT myelodysplastic syndrome (MDS), and only two relapsed post-HSCT. Forty two percent (25/59) had abnormal cytogenetics pre-HSCT; amongst 23 patients with serial pre/post bone marrow cytogenetic evaluations, 19 had established normal cytogenetics post-HSCT. Forty five percent (19/42) of MRD/URD patients received Tacro/MTX for GVHD prophylaxis, and 31.5% (6/19) developed grade III-IV acute GVHD (aGVHD). In contrast, 55% (23/42) MRD/URD patients received PT/Cy for GVHD prophylaxis, and none developed grade III-IV aGVHD (p 0.0052). Five percent (1/17) of HRD recipients developed grade III-IV aGVHD. Forty two percent (8/19) of MRD/URD patients who received post-transplant Tacro/MTX had chronic GVHD, 4.3% (1/23) of MRD/URD patients who received PT/Cy developed cGVHD, and 5.8% (1/17) of HRD patients developed cGVHD within the first 2 years post-transplant. Conclusions: Allogeneic HSCT using a busulfan-based regimen in GATA2 deficiency results in nearly a 90% disease-free survival with long-term immune reconstitution. The use of PT-Cy reduced the risk of severe aGVHD and cGVHD and did not increase the risk of relapse or progression of MDS/AML. With earlier recognition of the disease, and the use of PT/Cy more broadly, these results are expected to continue to improve. Disclosures Notarangelo: NIAID, NIH: Research Funding.

In Vivo Gene Therapy for Canine SCID-X1 Using Cocal-Pseudotyped Lentiviral Vector.

Hematopoietic stem and progenitor cell (HSPC)-based ex vivo gene therapy has demonstrated clinical success for X-linked severe combined immunodeficiency (SCID-X1) patients who lack a suitable donor for HSPC transplantation. Nevertheless, this form of treatment is associated with an increased risk of infectious disease complications and genotoxicity mainly due to the conditioning regimen. In addition, ex vivo gene therapy approaches require sophisticated facilities to manufacture gene-modified cells and to care for the patients after chemotherapy. Considering these impediments, we have developed an in vivo gene therapy approach to treat canine SCID-X1 after HSPC mobilization and systemic delivery of the therapeutic vector. Here, we investigated the use of the cocal envelope to pseudotype a lentiviral (LV) vector expressing a functional gammaC gene. The cocal envelope is resistant to serum inactivation compared with the commonly used vesicular stomatitis virus envelope glycoprotein (VSV-G) envelope and thus well suited for systemic delivery. Two SCID-X1 neonatal canines treated with this approach achieved long-term therapeutic immune reconstitution with no prior conditioning. Therapeutic levels of gene-corrected CD3+ T cells were demonstrated for at least 16 months, and all other correlates of T cell functionality were within normal range. Retroviral integration-site analysis demonstrated polyclonal T cell reconstitution. Comparative analysis of integration profiles of foamy viral (FV) vector and cocal LV vector after in vivo gene therapy found distinct integration-site patterns. These data demonstrate that clinically relevant and durable correction of canine SCID-X1 can be achieved with in vivo delivery of cocal LV. Since manufacturing of cocal LV is similar to VSV-G LV, this approach is easily translatable to a clinical setting, thus providing for a highly portable and accessible gene therapy platform for SCID-X1.

Adult and Cord Blood-Derived High-Affinity gB-CAR-T Cells Effectively React Against Human Cytomegalovirus Infections.

Human cytomegalovirus (HCMV) reactivations are associated with lower overall survival after transplantations. Adoptive transfer of HCMV-reactive expanded or selected T cells can be applied as a compassionate use, but requires that the human leukocyte antigen-matched donor provides memory cells against HCMV. To overcome this, we developed engineered T cells expressing chimeric antigen receptors (CARs) targeted against the HCMV glycoprotein B (gB) expressed upon viral reactivation. Single-chain variable fragments (scFvs) derived from a human high-affinity gB-specific neutralizing monoclonal antibody (SM5-1) were fused to CARs with 4-1BB (BBL) or CD28 (28S) costimulatory domains and subcloned into retroviral vectors. CD4+ and CD8+ T cells obtained from HCMV-seronegative adult blood or cord blood (CB) transduced with the vectors efficiently expressed the gB-CARs. The specificity and potency of gB-CAR-T cells were demonstrated and compared in vitro using the following: 293T cells expressing gB, and with mesenchymal stem cells infected with a HCMV TB40 strain expressing Gaussia luciferase (HCMV/GLuc). BBL-gB-CAR-T cells generated with adult or CB demonstrated significantly higher in vitro activation and cytotoxicity performance than 28-gB-CAR-T cells. Nod.Rag.Gamma (NRG) mice transplanted with human CB CD34+ cells with long-term human immune reconstitution were used to model HCMV/GLuc infection in vivo by optical imaging analyses. One week after administration, response to BBL-gB-CAR-T cell therapy was observed for 5/8 mice, defined by significant reduction of the bioluminescent signal in relation to untreated controls. Response to therapy was sporadically associated with CAR detection in spleen. Thus, exploring scFv derived from the high-affinity gB-antibody SM5-1 and the 4-1BB signaling domain for CAR design enabled an in vitro high on-target effect and cytotoxicity and encouraging results in vivo. Therefore, gB-CAR-T cells can be a future clinical option for treatment of HCMV reactivations, particularly when memory T cells from the donors are not available.

Low T-Cell Receptor β ( ) Repertoire Diversity Early Post-Transplant for Severe Combined Immunodeficiency (SCID) Predicts Subsequent Failure of Immune Reconstitution

Introduction Development of a diverse T cell repertoire is associated with immune recovery following allogeneic hematopoietic cell transplantation (HCT) for SCID. Although CDR3 spectratyping and staining of Vβ families provide approximations of the diversity of T cell receptors, high-throughput sequencing (HTS) of the TRB repertoire allows precise evaluation of clonotype dynamics during immune reconstitution. We investigated whether longitudinal HTS analysis of TRB would accurately assess development of TCR repertoire diversity over time and reflect the quality of T cell reconstitution following HCT for SCID. We also studied the influence of SCID genotype, conditioning regimen, donor type, and occurrence of chronic graft versus host disease (cGVHD) on TCR diversity post HCT. We hypothesized that repertoire diversity may represent an early biomarker to predict long-term immune reconstitution vs. need for a second intervention. Methods HTS was used to study the composition and diversity of TRB repertoire of 27 SCID infants, pre-HCT and at 100 d, 6 and 12 mo and yearly post-treatment(s). Median time of follow-up was 48 mo. Subjects were part of a prospective study of SCID by the Primary Immunodeficiency Treatment Consortium. Equal amounts of total RNA extracted from peripheral blood was used as template to amplify TRB rearrangements. The VDJ statistics file (PAST program) was used to calculate a Shannon entropy (H) index to measure repertoire diversity. Results TRB sequence analysis of 27 SCID patients showed poor diversity at baseline, followed by improvement to normal complexity (H index >8.0) after HCT. Similar kinetics of development of TRB diversity were seen in patients with IL2RG, JAK3, and IL7R defects (n=16) as in those with RAG and Artemis defects (n=14). In the latter group, however, HCT with no conditioning or immune suppression only was associated with persistently lower diversity than HCT with conditioning (p 500 CD4+ T cells/ul or > 200 naive CD4+ T cells/ul at 6 mo post-HCT correlated with higher TRB diversity at 24 and 36 mo post-HCT (p Conclusions Analysis of TRB diversity permits a detailed assessment of development of a diverse T cell repertoire following cellular therapies for SCID and confirms the need for patient-tailored treatment strategies based on SCID genotype. We propose that analysis of TRB diversity at 100 d post-HCT may identify patients at risk for failure of sustained immune reconstitution, thus prompting a second intervention without delay.

National Institutes of Health-Defined Chronic Graft-vs.-Host Disease in Pediatric Hematopoietic Stem Cell Transplantation Patients Correlates With Parameters of Long-Term Immune Reconstitution.

Recent data revealed the importance of immune reconstitution (IR) for the evaluation of possible biomarkers in National Institutes of Health (NIH)–defined chronic graft-vs.-host disease (cGVHD) and its clinical aspects. In this large pediatric study (n = 146), we have analyzed whether cellular and humoral parameters of IR in the long-term follow-up (FU) with a special emphasis on B-cell reconstitution correlate with NIH-defined cGVHD criteria. HYPOTHESIS: we were especially interested in whether meaningful cGVHD biomarkers could be defined in a large pediatric cohort. We here demonstrate for the first time in a highly homogenous pediatric patient cohort that both cGVHD (n = 38) and its activity were associated with the perturbation of the B-cell compartment, including low frequencies of CD19+CD27+ memory B-cells and increased frequencies of circulating CD19+CD21low B-cells, a well-known hyperactivated B-cell subset frequently found elevated in chronic infection and autoimmunity. Notably, resolution of cGVHD correlated with expansion of CD19+CD27+ memory B-cells and normalization of CD19+CD21low B-cell frequencies. Moreover, we found that the severity of cGVHD had an impact on parameters of IR and that severe cGVHD was associated with increased CD19+CD21low B-cell frequencies. When comparing the clinical characteristics of the active and non-active cGVHD patients (in detail at time of analyses), we found a correlation between activity and a higher overall severity of cGVHD, which means that in the active cGVHD patient group were more patients with a higher disease burden of cGVHD—despite similar risk profiles for cGVHD. Our data also provide solid evidence that the time point of analysis regarding both hematopoietic stem cell transplantation (HSCT) FU and cGVHD disease activity may be of critical importance for the detailed investigation of pediatric cohorts. Finally, we have proven that the differences in risk factors and patterns of IR, with cGVHD as its main confounding factor, between malignant and non-malignant diseases, are important to be considered in future studies aiming at identification of novel biomarkers for cGVHD.

Long-Term Thymic Function and Reconstitution of the T Cell Compartment after T Cell-Replete Haplo-Identical Allografting

INTRODUCTION Post-transplant cyclophosphamide (PTCY) has expanded the application of haploidentical stem cell transplantation (haplo-HSCT). Thymic function may play a pivotal role in long-term clinical outcomes. OBJECTIVES To evaluate the kinetics of long-term immune thymus-dependent reconstitution after PTCY haplo-HSCT. METHODS Twenty-nine patients (median age 53) underwent T-cell replete haplo-HSCT with PTCY. Blood samples were collected before conditioning and at 1, 3, 6, 12, 18, 24 months after transplantation. Analyses of CD4 and CD8 T-cell subsets by flow-cytometry were correlated by generalized linear models with Real-Time PCR quantification of signal joint T-cell receptor excision DNA circles (sjTRECs), specific marker of naive T-cells thymopoiesis. A) Naive; b) central; c) memory; and d) revertant CD4 and CD8 T-cells were defined as follows: a) CD45RA+CD62L+; b) CD45RO+CD62L+; c) CD45RO+CD27-; and d) CD45RA+/45RO+, respectively. SjTRECs real-time PCR was performed on genomic DNA (100 ng) extracted from sorted CD4 and CD8 T-cells. RESULTS Following PTCY induced T-cell depletion, a constant gradual increase in absolute numbers of all CD4 and CD8 T cell subsets and of sjTRECs copies from the first month up to two years post-transplant was observed (Figure 1). Overall, at two years, CD4 and CD8 T-cell levels and sjTRECs levels were however lower than those observed in healthy donors. sjTRECs kinetics was associated with the increase in naive T-cells (overall, p CONCLUSIONS Active thymic function despite age-dependent involution, substantially contributes to T-cell reconstitution after haplo-HSCT. Chronic GVHD and older age are however significantly correlated with lower thymic activity. Overall, lower production of sjTRECs after haplo-HSCT as compared after HLA identical sibling HSCT may partly be due to a higher degree of “mismatching” of MHC molecules during thymic re-education.

Cord Blood IL-15 Activated NK Cells Increase Cord Blood CD34+CD133+CD45lo Cell Function through IFN-y Production and Direct Cell Contact

Delayed engraftment following cord blood (CB) transplantation remains a significant challenge. While cell dose is clearly limiting, CB derived hematopoietic stem cells (HSCs) also exhibit a deficit in homing and engraftment. IL-15 activated natural killer (NK) cells have been shown as potential promoters of homing and engraftment of CB HSCs. However, the role of NK cells and their underlying mechanisms in promoting CB HSCs requires further study. Here, we explore the effect of IL-15 activated CB NK cells on the functional properties of CB CD34+CD133+CD45lo HSCs. In addition, we define the mechanistic interaction between NK cells and HSCs that may increase CB-HSC engraftment and improve patient outcome post-HSC transplantation.We first determined whether IL-15 activated NK cells could improve HSC function in vitro. Purified CD56+CD3- NK cells from CB were stimulated overnight with IL-15 and cultured at a 1:5 cell ratio with autologous purified CD34+CD133+CD45lo cells (CB HSCs). IL-15 activated NK cells increased CXCR4 expression on CB HSCs when compared to cultures with resting NK cells or CB HSCS alone. As HSCs must migrate to the bone marrow in order to engraft and facilitate long-term immune reconstitution, we next assessed whether NK cells also impact on HSC migration, clonogenicity and proliferation. We found that elevated levels of CXCR4 on CB HSCs cultured with activated NK cells also translated into enhanced chemotaxis towards SDF-1α in vitro. IL-15 activated CB NK cells also increased CB HSC clonogenicity as evaluated by short-term in vitro cultures. The effect of activated NK cells on the clonogenic capacity of CB HSCs was cell dose dependent with the highest effect observed at a ratio of 1:10. To study CB HSC proliferation, CFSE stained CD34+CD133+CD45lo cells were cultured either alone, with resting NK cells or IL-15 activated NK cells. Cultures with IL-15 activated NK cells significantly increased CB HSC proliferation when compared to cultures with resting NK cells or CB HSCs alone [median percentage of proliferating CB-HSCs; 38.4% (34%-44.6%) vs. 46.7% (36%-53.4%) vs. 69% (59.6%-78.5%)]. Moreover, following the ability of IL-15 activated NK cells to upregulate CB HSC proliferation, we investigated whether CB HSCs still retained their long-term engraftment potential. We found that proliferating CB HSCs still recalled both their short-term and long term clonogenic capacity as evaluated by CFU assays and cobblestone cultures followed by long-term culture (LTC-IC) respectively. Finally, we demonstrated that IL-15 activated NK cells also possessed the ability to activate pAkt/pErk and pStat3. As pAkt/pErk and pStat3 are key mediators of cell survival proliferation, our findings identify that NK cells may promote the survival and proliferation of CB HSCs through activating pAkt/pErk and pStat3. These data suggest that IL-15 activated NK cells from CB are endowed with properties to promote the functional profile of CB HSCs that contribute to improved engraftment.To further understand the underlying mechanisms through which IL-15 activated NK cells exert their ability to upregulate the functional profile of CB HSCs, we used antibody blockade experiments. We showed that the ability of NK cells to increase CXCR4 expression on CB HSCs was mediated via the provision of IFN-γ, but not TNF-α or TNF-β. Whereas, the effect of NK cells on CB HSC function studied through clonogenicity, proliferation and signalling studies was only partially dependent on IFN-γ production by IL-15 activated NK cells. Using transwell experiments, we further determined that the ability of activated NK cells to upregulate CB HSC function is also partly dependent on direct NK cell/HSC cell contact. Subsequently, we found that the addition of blocking antibody against 2B4 in cultures containing IL-15 activated NK cells and CB HSC partially reversed the ability of NK cells to increase the clonogenic capacity, proliferation and Akt/Erk and Stat3 signalling of CB HSCs. Thus, the ability of IL-15 activated NK cells to increase the functional profile of CB HSCs depends on IFN-γ production and cell-cell contact involving 2B4.Our combined studies demonstrate a novel effect of IL-15 activated CB NK cells and their key factors as potential mediators of stem cell homing and engraftment, which could be utilized to develop strategies that will benefit all patients with haematological malignancies and improve CB transplantation. DisclosuresNo relevant conflicts of interest to declare.

Long-term immune reconstitution after matched unrelated hematopoietic stem cell transplantation for immunodeficiency

Long-term immune reconstitution after matched unrelated hematopoietic stem cell transplantation for immunodeficiency

Immunological correlates of favorable long-term clinical outcome in multiple sclerosis patients after autologous hematopoietic stem cell transplantation

High dose immunosuppression followed by autologous hematopoietic stem cell transplantation (AHSCT) induces prolonged clinical remission in multiple sclerosis (MS) patients. However, how patient immune profiles are associated with clinical outcomes has not yet been completely elucidated. In this study, 37 MS patients were assessed for neurological outcomes, thymic function and long-term immune reconstitution after AHSCT. Patients were followed for a mean (SD) of 68.5 (13.9) months post-transplantation and were retrospectively clustered into progression- and non-progression groups, based on Expanded Disease Status Scale (EDSS) outcomes at last visit. After AHSCT, both patient groups presented increased regulatory T-cell subset counts, early expansion of central- and effector-memory CD8+T-cells and late thymic reactivation. However, the non-progression group presented early expansion of PD-1+CD8+T-cells and of PD-1-expressing CD19+ B-cells. Here, we suggest that along with increased numbers of regulatory T-cell subsets, PD-1 inhibitory signaling is one possible immunoregulatory mechanism by which AHSCT restores immune tolerance in MS patients.

476. No GVHD, but Human Inflammatory “Cytokine Storm” and Mouse Macrophage Activation Upon Accelerated Development of Human CD4+ Effector Memory T Cells in Long-Term Humanized Mice

Non clinical assessment of immunotoxicity of human cellular products in vivo is limited by the lack of availability of pharm-tox models enabling a full range of human innate and adaptive responses. We previously demonstrated improved development of human adaptive T and B cell responses in mice receiving human hematopoietic stem cell transplantation (HSCT) in combination with prime/boost immunization with induced dendritic cells (iDCs), i.e. monocytes derived the stem cell donor reprogramed with a lentiviral vector for co-expression of GM-CSF, IFN-alpha and the CMV antigen pp65 that self differentiate into DCs in vivo (Salguero et al, 2014 J. Immunology; Daenthanasanmak et al, 2015 Mol. Ther. Meth.). For these previous studies, mice were kept for 6-10 weeks after iDC administration, and we did not observe signs of tumorigenicity or graft-versus-host disease (GVHD). After consultation with the German regulatory authorities, we were requested to perform pharm-tox analyses 26 weeks after iDC immunization. We report here a pilot-feasibility study with humanized mice showing long-term (more than 33-36 weeks after HSCT) robust immune reconstitution with human T cells (control mice, n=2 Vs. iDC-immunized mice, n=6). Longitudinal analyses of human immune reconstitution in blood samples confirmed accelerated development of CD4+ effector memory T cells 5-10 weeks after iDC immunization. No weight loss, GVHD or malignancies were observed after iDC immunization. 3/6 immunized mice developed skin erythema and inflammation around 23 weeks after iDC immunization and were sacrificed earlier for analyses. Analyses of all mice were performed at autopsy by macroscopic observations, H&E histopathology, immunohistochemistry and flow cytometry of target organs. Positivity for anti-human nuclei antibody, anti-human HLA-DR and anti-human CD3 leucocytes were detected in several tissues (spleen, bone marrow, brain, lungs, skin, eyelids, liver, and kidneys), confirming the long-term persistency of human T cells in the humanized mice. In inflamed skin, abundant human CD4+ T cells were localized in the basal layers of the epidermis and hair follicles in the dermis. Human macrophages (CD68+) could be detected in several tissues, but were rare in inflamed skin. However, in the inflamed dermis areas of ulceration, mouse macrophages (F4/80+) were observed frequently with strongly positive cytoplasm. Mice with ulcerations had significantly higher levels of several human inflammatory cytokines in plasma (IFN-gamma, IL-6, GM-CSF and IL-8) than immunized mice with no skin inflammation or control mice. In summary, a long-term pilot study exploring a hybrid human-mouse model system was feasible and iDCs accelerated mature human T cell reconstitution with no signs of tumorigenicity or GVHD. However, accumulation of human CD4+ memory T cells and activated mouse macrophages in inflamed skin was associated with elevated levels of human inflammatory cytokines resembling a “cytokine storm”.

284. Long-Term Therapeutic Immune Reconstitution in XSCID Canine Model via In Vivo Foamy Virus Delivery of Common Gamma Chain

X-linked combined immunodeficiency disease (XSCID) is caused by mutation in the common gamma chain, γC (interleukin-2 receptor subunit gamma, IL2RG) in both humans and canines. It is characterized by the inability of T-cell development leading to absence of T-cells in peripheral blood, lack of T-cell mediated immune response, low IgA and IgG levels, and early infant mortality. In the 1990s, human XSCID clinical trials utilizing gamma-retroviral vectors to deliver the IL2RG gene caused leukemia in 5 out of 20 patients due to vector integration in or near proto-oncogenes. Recent studies showed Foamy virus based vectors as an excellent alternative for in vivo gene-therapy because it is non-pathogenic in humans while exhibiting increased serum stability and favorable integration pattern. Previously, we have demonstrated CD3+ T-cell reconstitution in the canine model via intravenous injection of foamy virus expressing human elongation factor-1 alpha promoter (Ef1α)-yC. Unfortunately, the treated animals contained a low number of gene corrected progenitors at a sub-therapeutic level. Here, we achieved long-term therapeutic immune-reconstitution by intravenous delivery of a human phosphoglycerate kinase promoter (Pgk)-mediated γC foamy viral vector into XSCID neonatal canines. Long-term (2 years) post-injection follow-up demonstrated therapeutic levels of CD3+ T-cell expansion. Within the T-cell population, gene correction with Pgk-γC stabilized at ~80%. We validated T-cell functionality by using spectratyping analysis, which exhibited a diverse repertoire of receptor gene rearrangement. Retroviral integration site analysis (RIS) indicated polyclonal contribution to the reconstituted T-cells. Immunoglobulin ELISA assays showed that IgA and IgG levels in peripheral blood are comparable to normal healthy controls. We immunized the gene-corrected canine recipients with bacteriophage ϕx174 and confirmed production of specific IgG antibodies, showing the ability for isotype switching in B-lymphocytes. Currently, the gene-corrected canines exhibit comparable health and physical attributes to normal controls. Furthermore, semen from the gene-corrected male canine was used via artificial insemination to produce a litter of viable offsprings. In summary, our data demonstrate that Pgk-γC foamy viral vector delivered long-term therapeutic gene correction in a large-animal model for XSCID gene therapy. Most importantly, these results indicate that in vivo Pgk-γC foamy vector administration is a viable option for long-term immune reconstitution in future XSCID human clinical trials.

Gene Therapy for X-Linked Severe Combined Immunodeficiency: Where Do We Stand?

More than 20 years ago, X-linked severe combined immunodeficiency (SCID-X1) appeared to be the best condition to test the feasibility of hematopoietic stem cell gene therapy. The seminal SCID-X1 clinical studies, based on first-generation gammaretroviral vectors, demonstrated good long-term immune reconstitution in most treated patients despite the occurrence of vector-related leukemia in a few of them. This gene therapy has successfully enabled correction of the T cell defect. Natural killer and B cell defects were only partially restored, most likely due to the absence of a conditioning regimen. The success of these pioneering trials paved the way for the extension of gene-based treatment to many other diseases of the hematopoietic system, but the unfortunate serious adverse events led to extensive investigations to define the retrovirus integration profiles. This review puts into perspective the clinical experience of gene therapy for SCID-X1, with the development and implementation of new generations of safer vectors such as self-inactivating gammaretroviral or lentiviral vectors as well as major advances in integrome knowledge.

121. Targeted Gene Therapy in the Treatment of X-Linked Hyper IgM Syndrome

X-linked hyper-IgM syndrome results in absent IgG, IgA, IgE and normal/elevated IgM due to defects in the CD40 ligand gene. HSCT is the only curative modality, but it carries significant risks, suggesting the need for improved methods of treatment. TALENs or CRISPRs, combined with the effective delivery of a homologous donor sequence containing normal CD40L DNA, will allow for targeted integration and provide physiologic expression of the endogenous CD40L gene to provide long-term immune reconstitution. TALENs targeting the 5′ UTR of the CD40L gene lead to allelic disruption of up to 31%at the target locus inK562 cells. In order to evaluate the capacity for targeted integration of a cassette at the cut site, cells were electroporated with the TALEN pair and a donor molecule with a promoterless GFP reporter gene flanked by homology arms. Expression of the GFP reporter was evaluated in Jurkat cells, with up to 10 % detected by flow cytometry and increasing to 22 % upon PHA activation. In addition, CRISPRs targeting a patient-specific mutation in intron 3 achieved >50 % gene disruption in K562 cells. Co-electroporation with a donormodified to contain a unique restriction enzyme site demonstrated site-specific gene integration. Site-specific modification at CD40L is achievable and physiologic expression of the endogenous CD40L gene could provide a viable therapy for XHIM.

The successful application of a national peer advisory committee for physicians who provide salvage regimens to heavily antiretroviral-experienced patients in mexican human immunodeficiency virus clinics.

Designing optimal antiretroviral (ARV) salvage regimens for multiclass drug-resistant, human immunodeficiency virus (HIV)-infected patients demands specific clinical skills. Our aim was to assess the virologic and immunologic effects of the treatment recommendations drafted by a peer advisory board to physicians caring for heavily ARV-experienced patients. We conducted a nationwide, HIV clinic-based, cohort study in Mexico. Adults infected with HIV were assessed for a median of 33 months (interquartile range [IQR] = 22-43 months). These patients had experienced the virologic failure of at least 2 prior ARV regimens and had detectable viremia while currently being treated; their physicians had received therapeutic advice, by a panel of experts, regarding the ARV salvage regimen. The primary endpoint was the incidence of loss of virologic response (plasma HIV-RNA levels of <200 copies per mL, followed by levels above this threshold) during the follow-up assessment using an observed-failure competing risks regression analysis. A total of 611 patients were observed (median ARV therapy exposure = 10.5 years; median prior regimens = 4). The probabilities of virologic failure were 11.9%, 14.4%, 16.9%, and 19.4% at the 12-, 24-, 36-, and 48-month follow-up assessments, respectively. Of the 531 patients who achieved a confirmed plasma HIV-RNA level below 200 copies per mL, the median increase in blood CD4(+) T-cell count was 162 cells per mL (IQR = 45-304 cells per mL). In routine practice, a high rate of patients with extensive ARV experience, who received an optimized salvage regimen recommended by a peer advisory committee, achieved a long-term sustained virologic response and immune reconstitution.