Postdepletion Lymphocyte Reconstitution During Belatacept and Rapamycin Treatment in Kidney Transplant Recipients.

Belatacept is used to prevent allograft rejection but fails to do so in a sizable minority of patients due to inadequate control of costimulation-resistant T cells. In this study, we report control of costimulation-resistant rejection when belatacept was combined with perioperative alemtuzumab-mediated lymphocyte depletion and rapamycin. To assess the means by which the alemtuzumab, belatacept and rapamycin (ABR) regimen controls belatacept-resistant rejection, we studied 20 ABR-treated patients and characterized peripheral lymphocyte phenotype and functional responses to donor, third-party and viral antigens using flow cytometry, intracellular cytokine staining and carboxyfluorescein succinimidyl ester-based lymphocyte proliferation. Compared with conventional immunosuppression in 10 patients, lymphocyte depletion evoked substantial homeostatic lymphocyte activation balanced by regulatory T and B cell phenotypes. The reconstituted T cell repertoire was enriched for CD28(+) naïve cells, notably diminished in belatacept-resistant CD28(-) memory subsets and depleted of polyfunctional donor-specific T cells but able to respond to third-party and latent herpes viruses. B cell responses were similarly favorable, without alloantibody development and a reduction in memory subsets-changes not seen in conventionally treated patients. The ABR regimen uniquely altered the immune profile, producing a repertoire enriched for CD28(+) T cells, hyporesponsive to donor alloantigen and competent in its protective immune capabilities. The resulting repertoire was permissive for control of rejection with belatacept monotherapy.

CD57(+) CD4 T Cells Underlie Belatacept-Resistant Allograft Rejection.

Belatacept is a B7-specific fusion protein used to prevent allograft rejection by blocking T cell costimulation. Generally efficacious, it fails to prevent acute rejection in a sizable minority of patients. In experimental models, memory T cells mediate costimulation blockade-resistant rejection (CoBRR), but this remains undefined in humans. To explore relationships between individual patients' immune cell phenotypes and CoBRR, we studied patients receiving belatacept or conventional calcineurin inhibitor-based immunosuppression. We identified a population of CD57(+) PD1(-) CD4 T cells present prior to transplantation that correlated with CoBRR. Contrary to data recognizing CD57 as a marker of senescence on CD8 T cells, we discovered a nonsenescent, cytolytic phenotype associated with CD57 on CD4 T cells. Moreover, CD57(+) CD4 T cells expressed high levels of adhesion molecules implicated in experimental CoBRR, were CD28(-) , expressed a transcriptional phenotype broadly defining allograft rejection and were shown to be present in rejecting human kidney allografts. These data implicate CD57(+) CD4 T cells in clinical CoBRR. If prospectively validated, this characteristic could identify patients at higher risk for acute rejection on belatacept-based therapy.

Studies Introducing Costimulation Blockade for Vascularized Composite Allografts in Nonhuman Primates.

Vascularized composite allografts (VCAs) are technically feasible. Similar to other organ transplants, VCAs are hampered by the toxicity and incomplete efficacy associated with conventional immunosuppression. Complications attributable to calcineurin inhibitors remain prevalent in the clinical cases reported to date, and these loom particularly large given the nonlifesaving nature of VCAs. Additionally, acute rejection remains almost ubiquitous, albeit controllable with current agents. Costimulation blockade offers the potential to provide prophylaxis from rejection without the adverse consequences of calcineurin-based regimens. In this study, we used a nonhuman-primate model of VCA in conjunction with immunosuppressive regimens containing combinations of B7-specific costimulation blockade with and without adhesion blockade with LFA3-Ig to determine what adjunctive role these agents could play in VCA transplantation when combined with more conventional agents. Compared to tacrolimus, the addition of belatacept improved rejection free allograft survival. The combination with LFA3-Ig reduced CD2(hi) memory T cells, however did not provide additional protection against allograft rejection and hindered protective immunity. Histology paralleled clinical histopathology and Banff grading. These data provide the basis for the study of costimulation blockade in VCA in a relevant preclinical model.

Superiority of rapamycin over tacrolimus in preserving nonhuman primate Treg half-life and phenotype after adoptive transfer.

Many critical issues remain concerning how best to deploy adoptive regulatory T cell (Treg) immunotherapy to the clinic. These include a determination of their pharmacokinetic characteristics, their optimal dose, their phenotypic stability and the best therapies with which to pair Tregs. By performing a CFSE-labeled autologous Treg pulse experiment, we determined that the accessible peripheral blood Treg pool in rhesus macaques is quite large (75 ± 11 × 10(6) Tregs/kg). Pharmacokinetic analysis revealed that Tregs have two phases of elimination: an α phase, with a T1/2 in the peripheral blood of 32.4 ± 11.3 h and a ß phase with a T1/2 of 120.4 ± 19.7 h. In addition to their short initial half-life, Tregs underwent rapid phenotypic shifts after infusion, with significant loss of both CD25 and FoxP3 by day +6. While tacrolimus stabilized CD25 expression, it did not improve T1/2 , nor mitigate the loss of FoxP3. In contrast, rapamycin significantly stabilized both CD25 and FoxP3, and supported an increased half-life, with an α phase of 67.7 ± 6.9 h and a ß phase of 252.1 ± 54.9 h. These results suggest that rapamycin may be a necessary addition to Treg immunotherapy, and that tacrolimus may be deleterious to Treg integrity posttransfer.

Regulatory T cells exhibit decreased proliferation but enhanced suppression after pulsing with sirolimus.

Although regulatory T cells (Tregs) suppress allo-immunity, difficulties in their large-scale production and in maintaining their suppressive function after expansion have thus far limited their clinical applicability. Here we have used our nonhuman primate model to demonstrate that significant ex vivo Treg expansion with potent suppressive capacity can be achieved and that Treg suppressive capacity can be further enhanced by their exposure to a short pulse of sirolimus. Both unpulsed and sirolimus-pulsed Tregs (SPTs) are capable of inhibiting proliferation of multiple T cell subpopulations, including CD4(+) and CD8(+) T cells, as well as antigen-experienced CD28(+) CD95(+) memory and CD28(-) CD95(+) effector subpopulations. We further show that Tregs can be combined in vitro with CTLA4-Ig (belatacept) to lead to enhanced inhibition of allo-proliferation. SPTs undergo less proliferation in a mixed lymphocyte reaction (MLR) when compared with unpulsed Tregs, suggesting that Treg-mediated suppression may be inversely related to their proliferative capacity. SPTs also display increased expression of CD25 and CTLA4, implicating signaling through these molecules in their enhanced function. Our results suggest that the creation of SPTs may provide a novel avenue to enhance Treg-based suppression of allo-immunity, in a manner amenable to large-scale ex vivo expansion and combinatorial therapy with novel, costimulation blockade-based immunosuppression strategies.

CD40 blockade combines with CTLA4Ig and sirolimus to produce mixed chimerism in an MHC-defined rhesus macaque transplant model.

In murine models, T-cell costimulation blockade of the CD28:B7 and CD154:CD40 pathways synergistically promotes immune tolerance after transplantation. While CD28 blockade has been successfully translated to the clinic, translation of blockade of the CD154:CD40 pathway has been less successful, in large part due to thromboembolic complications associated with anti-CD154 antibodies. Translation of CD40 blockade has also been slow, in part due to the fact that synergy between CD40 blockade and CD28 blockade had not yet been demonstrated in either primate models or humans. Here we show that a novel, nondepleting CD40 monoclonal antibody, 3A8, can combine with combined CTLA4Ig and sirolimus in a well-established primate bone marrow chimerism-induction model. Prolonged engraftment required the presence of all three agents during maintenance therapy, and resulted in graft acceptance for the duration of immunosuppressive treatment, with rejection resulting upon immunosuppression withdrawal. Flow cytometric analysis revealed that upregulation of CD95 expression on both CD4+ and CD8+ T cells correlated with rejection, suggesting that CD95 may be a robust biomarker of graft loss. These results are the first to demonstrate prolonged chimerism in primates treated with CD28/mTOR blockade and nondepletional CD40 blockade, and support further investigation of combined costimulation blockade targeting the CD28 and CD40 pathways.

Evidence for kidney rejection after combined bone marrow and renal transplantation despite ongoing whole-blood chimerism in rhesus macaques.

Although there is evidence linking hematopoietic chimerism induction and solid organ transplant tolerance, the mechanistic requirements for chimerism-induced tolerance are not clearly elucidated. To address this, we used an MHC-defined primate model to determine the impact of impermanent, T cell-poor, mixed-chimerism on renal allograft survival. We compared two cohorts: one receiving a bone marrow and renal transplant ("BMT/renal") and one receiving only a renal transplant. Both cohorts received maintenance immunosuppression with CD28/CD40-directed costimulation blockade and sirolimus. As previously demonstrated, this transplant strategy consistently induced compartmentalized donor chimerism, (significant whole-blood chimerism, lacking T cell chimerism). This chimerism was not sufficient to prolong renal allograft acceptance: the BMT/renal mean survival time (MST, 76 days) was not significantly different than the renal transplant alone MST (85 days, p = 0.46), with histopathology documenting T cell mediated rejection. Flow cytometric analysis revealed significant enrichment for CD28-/CD95+ CD4+ and CD8+ Tem cells in the rejected kidney, suggesting a link between CD28-negative Tem and costimulation blockade-resistant rejection. These results suggest that in some settings, transient T cell-poor chimerism is not sufficient to induce tolerance to a concurrently placed renal allograft and that the presence of this chimerism per se is not an independent biomarker to identify tolerance.

Selective targeting of human alloresponsive CD8+ effector memory T cells based on CD2 expression.

Costimulation blockade (CoB), specifically CD28/B7 inhibition with belatacept, is an emerging clinical replacement for calcineurin inhibitor-based immunosuppression in allotransplantation. However, there is accumulating evidence that belatacept incompletely controls alloreactive T cells that lose CD28 expression during terminal differentiation. We have recently shown that the CD2-specific fusion protein alefacept controls costimulation blockade-resistant allograft rejection in nonhuman primates. Here, we have investigated the relationship between human alloreactive T cells, costimulation blockade sensitivity and CD2 expression to determine whether these findings warrant potential clinical translation. Using polychromatic flow cytometry, we found that CD8(+) effector memory T cells are distinctly high CD2 and low CD28 expressors. Alloresponsive CD8(+) CD2(hi) CD28(-) T cells contained the highest proportion of cells with polyfunctional cytokine (IFNγ, TNF and IL-2) and cytotoxic effector molecule (CD107a and granzyme B) expression capability. Treatment with belatacept in vitro incompletely attenuated allospecific proliferation, but alefacept inhibited belatacept-resistant proliferation. These results suggest that highly alloreactive effector T cells exert their late stage functions without reliance on ongoing CD28/B7 costimulation. Their high CD2 expression increases their susceptibility to alefacept. These studies combined with in vivo nonhuman primate data provide a rationale for translation of an immunosuppression regimen pairing alefacept and belatacept to human renal transplantation.

An MHC-defined primate model reveals significant rejection of bone marrow after mixed chimerism induction despite full MHC matching.

In murine models, mixed hematopoietic chimerism induction leads to robust immune tolerance. However, translation to primates and to patients has been difficult. In this study, we used a novel MHC-defined rhesus macaque model to examine the impact of MHC matching on the stability of costimulation blockade-/sirolimus-mediated chimerism, and to probe possible mechanisms of bone marrow rejection after nonmyeloablative transplant. Using busulfan-based pretransplant preparation and maintenance immunosuppression with sirolimus, as well as CD28 and CD154 blockade, all recipients demonstrated donor engraftment after transplant. However, the mixed chimerism that resulted was compartmentalized, with recipients demonstrating significantly higher whole blood chimerism compared to T cell chimerism. Thus, the vast majority of T cells presenting posttransplant were recipient-rather than donor-derived. Surprisingly, even in MHC-matched transplants, rejection of donor hematopoiesis predominated after immunosuppression withdrawal. Weaning of immunosuppression was associated with a surge of antigen-experienced T cells, and transplant rejection was associated with the acquisition of donor-directed T cell alloreactivity. These results suggest that a reservoir of alloreactive cells was present despite prior costimulation blockade and sirolimus, and that the post-immunosuppression lymphocytic rebound may have lead to a phenotypic shift in these recipient T cells towards an activated, antigen-experienced phenotype, and ultimately, to transplant rejection.

Expanded nonhuman primate tregs exhibit a unique gene expression signature and potently downregulate alloimmune responses.

We have established two complementary strategies for purifying naturally occurring regulatory T cells (Tregs) from rhesus macaques in quantities that would be sufficient for use as an in vivo cellular therapeutic. The first strategy identified Tregs based on their being CD4+/CD25(bright). The second incorporated CD127, and purified Tregs based on their expression of CD4 and CD25 and their low expression of CD127. Using these purification strategies, we were able to purify as many as 1x10(6) Tregs from 120 cc of peripheral blood. Cultures of these cells with anti-CD3, anti-CD28 and IL-2 over 21 days yielded as much as a 450-fold expansion, ultimately producing as many as 4.7x10(8) Tregs. Expanded Treg cultures potently inhibited alloimmune proliferation as measured by a carboxyfluorescein succinimidyl ester- mixed lymphocyte reaction (CFSE-MLR) assay even at a 1:100 ratio with responder T cells. Furthermore, both responder-specific and third-party Tregs downregulated alloproliferation similarly. Both freshly isolated and cultured Tregs had gene expression signatures distinguishable from concurrently isolated bulk CD4+ T-cell populations, as measured by singleplex reverse transcriptase-polymerase chain reaction (RT-PCR) and gene array. Moreover, an overlapping yet distinct gene expression signature seen in freshly isolated compared to expanded Tregs identifies a subset of Treg genes likely to be functionally significant.

Lymphoid-associated antigen expression by acute myeloid leukemia.

The expression of lymphoid-associated antigens (LAA) on blasts in acute myeloid leukemia (AML) and myeloproliferative disorders in myeloid blast crisis (MPD/MBC) has often been used to establish a diagnosis of acute mixed lineage leukemia (AMLL). The purpose of this study was to determine the incidence of LAA expression in AML and MPD/MBC (Ly + AML); to assess lymphoid differentiation at the genomic level in Ly + AML; and to compare features of Ly + AML with AML and MPD/MBC lacking these antigens (Ly-AML). Seventy-four consecutive cases of AML and MPD/MBC were reviewed for blast morphology, TdT reactivity, and cytochemistry results. Blast immunophenotyping was performed by multiparameter flow cytometry. Acute myeloid leukemia was subtyped according to the FAB classification. Acute myeloid leukemia and MPD/MBC cases expressing one or more of the following antigens, CD2, CD3, CD5, CD7, CD19, or CD20, were considered to be Ly + AML. Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement studies were performed by Southern blot analysis using probes for JH, Jkappa, and JBI/BII. Sixteen of the 74 cases (22%) were identified as Ly + AML. Of these, the T-cell-associated markers CD7, CD2, and CD5 were expressed on 7(44%), 6(38%), and 4(25%) Ly + AML cases, respectively. The B-cell-associated markers CD19 and CD20 were expressed on two cases (13%) and one (6%) case, respectively. The FAB subtypes were similarly represented among Ly + AML and Ly-AML. Expression of LAA did not correlate with TdT positivity. In nine cases of Ly + AML (7 expressing T-cell-associated antigens and two expressing B-cell-associated antigens), Southern blot analysis revealed no Ig or TCR gene rearrangements. These results suggest that expression of CD2, CD5, and CD7 in otherwise straightforward AML should not be taken as evidence of lymphoid lineage commitment and does not warrant a diagnosis of AMLL.

CD117/CD34 expression in leukemic blasts.

CD117 is a transmembrane protein receptor encoded by the c-kit proto-oncogene. The CD117 ligand is stem cell factor, an important hematopoietic regulator. CD117 is present on approximately 4% of normal bone marrow mononuclear cells and in acute myelogenous leukemia (AML) and chronic myelogenous leukemia in myeloid blast crisis, but rarely in acute lymphoblastic leukemia (ALL). Initially viewed as a primitive myeloid marker, CD117 has been identified in all FAB subtypes of AML and may predict poor outcome. CD34, a primitive stem cell marker, may also predict poor outcome. The aim of this study was to examine the relationship between CD117 and CD34 expression on leukemic blasts and to determine whether CD117 is related to lymphoid-associated antigen (LAA) expression in AML. Consecutive bone marrow samples were studied from cases of AML (30 cases), myelodysplastic syndromes (MDS) (4 cases), myeloproliferative disorders in blast crisis (MPD-BC) (6 cases), and ALL (5 cases). Cases were diagnosed according to FAB criteria and included M0 (3 cases), M1 (2 cases), M2 (13 cases), M3 (1 case), M4 (6 cases), M5 (3 cases), M6 (1 case), AML NOS (1 case), RAEB (3 cases), and RAEB-T (1 case). CD117 and CD34 were analyzed by multiparameter flow cytometry. Blasts in 10 de novo AML samples were CD117+/CD34+ in 4 cases, CD117+/CD34-in 3 cases, CD117-/CD34+ in 1 case, and CD117-/ CD34- in 2 cases. Blasts in 20 cases of relapsed AML were CD117+/ CD34+ in 13 cases, CD117+/CD34- in 6 cases, and CD117-/CD34+ in 1 case. Blasts in MDS were CD117+/CD34+ in 3 cases, CD117-/ CD34+ in 1 case. Blasts in MPD-BC were CD117+/CD34+ in 4 cases, CD117-/CD34+ in 2 cases. Blasts in ALL were CD117+/CD34+ in 1 case, CD117-/CD34+ in 1 case, CD117-/CD34- in 3 cases. Of 26 cases of CD117+ AML, CD4 was expressed in 15 (58%) cases, CD7 in 7 (27%) cases, and CD2 in 2 (8%) cases. CD117/CD34 expression did not correlate with FAB subtype of AML. CD117 is borne on most leukemic blasts of myeloid origin (in this study, 87% of AML, 80% of MPD-myeloid BC, and 75% of MDS) and does not exclude expression of LAA. Although CD117 is a receptor for stem cell factor, its expression does not appear to correlate with CD34 positivity.

DNA index and ploidy distinguish normal human parathyroids from parathyroid adenomas and primary hyperplastic parathyroids.

BACKGROUND: The goal of this study was to identify factors that might aid in diagnosis and intraoperative management of hyperparathyroidism. METHODS: We analyzed biopsy specimens of 242 parathyroids from 159 patients by use of flow cytometry and image cytometry (ICM) for DNA index (DI), defined as the content of nuclear DNA compared with that expected for a DNA diploid standard, for proliferative index (PI), and for ploidy (diploid versus aneuploid or tetraploid). RESULTS: True normal and normal parathyroids from patients with solitary adenomas were uniformly diploid. Abnormal ploidy (aneuploidy or tetraploidy) was identified frequently in adenomas and occasionally in hyperplasias with the exception that multiple endocrine neoplasia (MEN) biopsy specimens were uniformly diploid. DI for adenomas was similar to that for hyperplasias, and DI of both was higher than for normal glands. ICM-DI correlated positively with flow cytometry-DI and patient age and inversely with serum parathyroid hormone. PI was relatively low in all groups but was higher for hyperplasias versus normal parathyroids from patients with solitary adenomas and MEN versus non-MEN. PI correlated inversely with patient age. CONCLUSIONS: DI by ICM differentiates normal from abnormal parathyroids. DI might influence extent of resection in two- and three-gland hyperplasia and selection of the most appropriate gland for autografting and cryopreservation in patients with four-gland hyperplasia.

NK cells mediate costimulation blockade-resistant rejection of allogeneic stem cells during nonmyeloablative transplantation.

Although T-cell CD28/CD40 costimulation blockade represents a powerful mechanism to promote immune tolerance during murine allotransplantation, it has not yet been successfully translated to clinical transplantation. We determined the impact of natural killer (NK) cells on costimulation blockade-resistant rejection of donor bone marrow. We found that NK cells represent a potent barrier to engraftment: host NK depletion led to increased donor stem cell survival, increased mixed hematopoietic chimerism and to engraftment of low doses of donor marrow (1 x 10(8)/kg) that were otherwise rejected. To understand the mechanisms of NK alloreactivity, we employed an in vivo NK-specific cytotoxicity assay. We found that an increased proportion of target cells were killed between days 2 and 8 after cell transfer, and that NK killing of parental targets was inducible: NK cells preprimed with allotargets were more efficient at their elimination upon reexposure. Finally, both transplant and in vivo NK-killing models were used to determine the contribution of LFA-1 to NK alloreactivity. Blockade of LFA-1 led to decreased NK-mediated killing, and increased alloengraftment. These results identify NK alloreactivity as an integral component to costimulation blockade-resistant rejection, and suggest that its inhibition may represent an important target in the clinical translation of tolerance-induction transplantation.

Characterization of Thy-1 (CDw90) expression in CD34+ acute leukemia.

Thy-1 (CDw90) is a phosphatidylinositol-anchored cell surface molecule which, when coexpressed with CD34 in normal human bone marrow, identifies a population of immature cells that includes putative hematopoietic stem cells. To date, the characterization of Thy-1 expression has been confined largely to normal tissues and cell lines. In this study, we evaluated the frequency and intensity of Thy-1 expression as defined by reactivity with the anti-Thy-1 antibody 5E10 in 38 cases of CD34+ acute leukemia (21 acute myelogenous leukemia [AML], 8 chronic myelogenous leukemia [CML] in blast crisis, and 9 acute lymphoblastic leukemia [ALL]). In 34 of 38 cases (89%) the CD34+ cells lacked expression of the Thy-1 antigen. High-density Thy-1 expression was found in 1 case of CML in lymphoid blast crisis, and low-density Thy-1 expression was identified on a portion of the leukemic cells in 2 cases of AML with myelodysplastic features, and 1 case of CML in myeloid blast crisis, suggesting a possible correlation between Thy-1 expression and certain instances of stem cell disorders such as CML and AML with dysplastic features. In contrast, the dissociation of Thy-1 and CD34 expression in the majority of acute leukemias studied suggests that the development of these leukemias occurs at a later stage than the hematopoietic stem cell. Characterization of Thy-1 expression in acute leukemia may eventually provide insights into the origin of the disease. In addition, separation of leukemic blasts from normal stem cells based on Thy-1 expression may prove useful in assessing residual disease, as well as in excluding leukemic blasts from stem cell preparations destined for autologous bone marrow or peripheral stem cell transplantation.

CD34+ progenitors and colony-forming units-granulocyte macrophage are recruited during large-volume leukapheresis and concentrated by counterflow centrifugal elutriation.

The recruitment of mononuclear cells (MNCs), colony-forming units-granulocyte macrophage (CFU-GM), lymphocyte subpopulations, and CD34+ progenitor cells was studied during large-volume (15-25 L blood processed) peripheral blood stem cell (PBSC) harvests. Normal donors (n = 13) underwent a 4-hour leukapheresis designed to maximize PBSC yield (blood flow rate, 85 mL/min). Mean (+/- SD) volume processed was 17.7 +/- 0.4 L, and yield was 2.4 +/- 0.7 x 10(10) white cells containing 99 percent MNCs and 1.3 mL red cells per L of blood processed. Postapheresis hematocrit, platelets, and MNCs were reduced from preapheresis values by 7, 35, and 23 percent, respectively (p < 0.05). In nine donors, the component was collected as four 1-hour samples, and culturing of CFU-GM and flow cytometric analysis of lymphocyte subpopulations and CD34+/HLA-DR+ cells were done in individual samples. Total CFU-GM were 2.4 +/- 1.4 x 10(6) (3.0 +/- 1.8 x 10(4) CFU-GM/kg) and lymphocytes were 20.8 x 10(9), with 75 percent CD3+ T cells, 10 percent CD19/CD20+ B cells, and 17 percent natural killer cells. A more than twofold increase in CFU-GM and CD34+ cells was noted over the course of the 4-hour procedure (p < 0.05). In four donors, the leukapheresis component underwent counterflow centrifugal elutriation (CCE), which separated it into four fractions in an attempt to concentrate CD34+ and CFU-GM progenitors and to deplete T-lymphocytes on a large scale. There was a 1.8-, 4.6-, 3.9-, and 0.32-fold increase in CFU-GM in the four fractions relative to the unseparated component.(ABSTRACT TRUNCATED AT 250 WORDS)