MLR Responses Research Articles

Altered Vestibular Balance Function in Combat Sport Athletes.

Combat sports pose a risk for accumulative injuries to the nervous system, yet fighters have remained an understudied population. Here, our purpose was to determine whether repetitive blows to the head have an effect on vestibular balance reflexes in combat sports athletes. We compared lower-limb muscle responses evoked with electrical vestibular stimuluation (EVS) between fighters (boxing/muay thai) and non-fighter controls. Each participant received stochastic vestibular stimulation (0-25 Hz, ±3 mA) over their mastoid processes while they stood relaxed with their head to the left or right. Surface electromyography was recorded from the medial gastrocnemius and soleus muscles bilaterally. Short and medium latency response (SLR/MLR) peaks were significantly delayed in the fighter group compared to controls. SLR and MLR peak amplitudes were also significantly lower in fighters. Fighter-estimated cumulative repetitive head impact (RHI) events demonstrated strong positive correlations with the timing of SLR and MLR peaks. Cumulative RHI events also negatively correlated with peak MLR amplitude and response gain at frequencies above 5 Hz. Our results provide evidence of a progressive vestibular impairment in combat sports athletes, potentially resulting from blows to the head accumulated in sparring practice and competitive bouts throughout their careers. Taken together, EVS-based vestibular assessments may provide a valuable clinical diagnostic tool and help better inform "return-to-play" and career-length decisions for not only combat sports athletes, but potentially other populations at risk of RHIs.

Neuromapping of the Capsuloligamentous Knee Joint Structures

PurposeTo investigate neuromuscular electromyographic response of the of the upper and lower leg muscles after the application of an intraoperative, isolated mechanical stimulus of the capsuloligamentous structures, including the anterior (ACL) and posterior cruciate ligaments (PCL), lateral (LM) and medial menisci (MM), plica mediopatellaris (PM), and Hoffa’s fat pat (HFP).MethodsThe electromyographic response of the upper and lower leg muscles (M. rectus femoris; M. vastus medialis; M. semitendinosus; M. biceps femoris; M. gastrocnemius lateralis) of 15 male patients were measured after an isolated mechanical stimulus of the capsuloligamentous structures during an arthroscopic intervention using a customized intraoperative setup. Target parameters were the short (SLR; <30 milliseconds) and medium latency responses (MLR; >30 milliseconds) after the mechanically-induced trigger.ResultsThe ACL, PCL, LM, and MM displayed high interindividual reproducibility of >76%. The MM was the only structure indicating both an SLR and MLR for all muscles. Although signals could be detected, there was no reproducibility in electromyographic signal activation for the HFP. The most rapid MLR was observed for the PM (quadriceps: 37 milliseconds).ConclusionsEach stimulated structure displayed an individual MLR response, which allowed us to create neuromapping combining the anatomical and quantitative representations of the individual muscular activation patterns after isolated mechanical stimulation of the capsuloligamentous knee joint structures, corroborating our hypothesis.Level of EvidenceDiagnostic - Level II.

Characterization of a mouse model of islet transplantation using MIN-6 cells

ABSTRACT Immortalized beta cells are an abundant source of insulin-producing cells. Although MIN-6 cells have similar characteristics as normal islets in vitro, the in vivo use of MIN-6 cells has not been fully described. This study characterizes in vivo mouse models of MIN-6 transplantation and rejection. Subcutaneous (sc) transplantation of MIN-6 cells in either Matrigel or HyStem-C hydrogels reduced blood sugars in nude mice and thus are good matrices for MIN-6 cells in vivo. NOD mice are good transplant recipients since they best rejected MIN-6 cells. MLR responses from BalbC, Black Webster, Swiss Black, C3H, and NOD mice correlated with mean blood glucose response suggesting the importance of allogeneic differences in the rejection of cells. Three days of cyclosporine administration caused no inhibition of MIN-6 cell rejection and 6 days resulted in a transient decrease in blood glucose, while daily administration inhibited rejection long term. Kinetic glucose tolerance (GTT) studies in nude mice demonstrated transplanted MIN-6 cells are close but not as effective as normal islets in controlling blood glucose and blood glucose set point for insulin release in MIN-6 cells decreases to hypoglycemic levels over time. To avoid hypoglycemia, the effect of MIN-6 cell irradiation was assessed. However, irradiation only delayed the development of hypoglycemia, not altering the final glucose set point for insulin release. In conclusion, we have characterized a mouse model for beta-cell transplantation using subcutaneous MIN-6 cells that can be used as a tool to study approaches to mitigate immune rejection.

Clonal Deletion and Anergy Play Dominant Role To Achieve Immune Tolerance After Reduced Intensity Unrelated Donor Cord Blood Transplantation (UCBT)

Abstract The mechanism of immune tolerance after successful hematopoietic stem cell transplantation is not fully understood. Both central (clonal deletion) and peripheral (anergy, Treg, Tr1) mechanisms are conceivable and may even coexist. In this study, we set up serial experiments to test for the presence and relative contribution of these mechanisms after HLA-mismatched UCBT in the GvH direction. Nine patients (age 1m to 9y) were transplanted and enrolled on a clinical trial (NCT01852370). Five immunocompetent patients were off immunosuppression therapy (IST) with no GVHD, while 4 had either limited or recently resolved GvHD and were still on IST when studied. Donor T cell chimerism was a median 97%. Purified T cells responses to host APCs were measured by MLR (3HTdR) and CTL reaction (Europium assay), along with cytokine secretion profiling (Bioplex). Host-reactive T cell clones were tracked with TCRVβ repertoire by ImmunoSEQ® (Adaptive Biotech). In tolerant patients, there was no significant MLR or CTL response towards host APC. Similarly, cytokine profiles revealed non-responsiveness while T cells responded vigorously to 3rd party APC by all assays. Recipient-specific T cell clones that were identified from infused UCB stimulated by host APC pre-UCBT became undetectable. There was no indication of Treg or Tr1 cell involvement in sustaining tolerance as deletion by IL-2 immunotoxin or IL-10R blockade was unable to “break” tolerance. However, low dose IL-2 (5U/mL) was able to restore some proliferation (S.I.=19; p=0.05) and IL-13, IFNγ, TNFα secretion, but in lesser degree in comparison to 3rd party responses (S.I.=112). In summary, clonal deletion and anergy contribute to immune tolerance after UCBT with deletion being dominant.

Functional consequences of TCR transgenic β-chain expression (TUM2P.908)

Abstract We have generated mouse strain expressing transgenic β-chain of TCR 1D1 (belonging to the Vβ6 family) on the genetic background B10.D2(R101) (KdIdDb). The repertoire of peripheral T lymphocytes in these mice contains 70-80% of T cells expressing transgenic β-chain and 20-30% of T cells expressing endogenous β-chains. Expression of transgenic β-chain resulted in T cells with phenotype CD44-CD62L+ characteristic of “naïve” T lymphocytes. On the contrary, CD8+ T lymphocytes expressing endogenous β-chains had surface phenotype of activated effectors CD44+CD62L-. In wild-type B10.D2(R101) mice, immunization with lymphoma EL4 (H-2b) cells induced a vigorous CTL response targeted to the H-2Kb molecule and resulted in full rejection of the tumor cells. In contrast, transgenic mice developed a compromised proliferative response in MLR and were unable to reject transplanted allogeneic EL4 cells. During the immune response to EL4 cells, mostly CD8+ T-lymphocytes with endogenous β-chains became CD44+CD62L- effectors in transgenic mice, whereas only a small part of the T-lymphocytes expressing transgenic β-chains became effectors. Effectors CD8+CD44+CD62L- expressing endogenous β-chains showed enhanced expression of PD-1 and contained increased percent of KLRG1-positive cells. The acquisition of these characteristic features was associated with immune selection of H-2Kb-negative EL4 cells and subsequent tumor escape.

Rapid Dendritic Cell Activation and Resistance to Allotolerance Induction in Anti-CD154-Treated Mice Receiving CD47-Deficient Donor-Specific Transfusion

CD47-SIRPα signaling plays an important role in regulating macrophage and dendritic cell (DC) activation. Here we investigated the role of CD47 expression on donor cells in tolerance induction by combined treatment with donor-specific transfusion (DST) plus anti-CD154 mAb in a mouse model of fully MHC-mismatched heart allotransplantation. The majority of BALB/c recipient mice that received anti-CD154 and CD47(+/+) B6 splenocytes (DST) showed indefinite donor heart survival (median survival time, MST > 150 days). Donor heart survival was improved in anti-CD154-treated BALB/c mice that received CD47(+/-) (MST = 90 days) or CD47(-/-) B6 DST (MST = 42 days) when compared to the nontreated (MST = 7 days) and anti-CD154 alone-treated (MST = 15 days) controls, but significantly reduced when compared to mice receiving anti-CD154 plus CD47(+/+) B6 DST. Recipient mice treated with anti-CD154 plus CD47(-/-) or CD47(+/-) DST also showed significantly increased antidonor, but not anti-third-party, MLR responses compared to those receiving anti-CD154 and CD47(+/+) DST. Furthermore, CD47(-/-) DST induced rapid activation of CD11c(hi)SIRPα(hi)CD8α(-) DCs via a mechanism independent of donor alloantigens. These results demonstrated that CD47 expression on donor cells is essential to the success of tolerance induction by combined therapy with DST and CD40/CD154 blockade.

Pharmacological preconditioning with adenosine A1 receptor agonist suppresses cellular immune response by an A2A receptor dependent mechanism

Under stressful conditions such as ischemia, sepsis, and severe trauma, adenosine levels are elevated and protect the tissue by interaction with G coupled receptors. In a model of peritonitis, we previously found that pharmacological preconditioning (PPC) of mice with a selective adenosine A1 receptor (A1R) agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA), induced the A2AR which reduces cytokine secretion and leukocyte recruitment. In our present study we determined whether mice PPC will moderate cellular immune response by the same mechanism. Similar to the effect on inflammation, PPC reduced the response to lymphocyte mitogens and allogeneic MLR response. The inhibitory effect of PPC on the immune response was A1R and A2AR dependent as illustrated by experiments with antagonists of these receptors and mice with knock down (KO) receptors. In MLR with PPC splenocytes we found reduced levels of pro-inflammatory cytokines (IFN-γ, IL-15, TNF-α) and elevation of IL-10, as well as elevation of regulatory T-cell. Our data indicate that PPC is able to remarkably suppress cellular immune response due to the sensitization A2AR. This effect of PPC sheds light on the protective role of adenosine in ischemic preconditioning and makes this treatment candidate for the prevention of graft rejection.

Aging effects on posture-related modulation of stretch reflex excitability in the ankle muscles in humans

The purpose of this study was to examine the effects of aging on posture-related changes of the stretch reflex excitability in the ankle extensor, soleus (SOL), and flexor, tibialis anterior (TA) muscles. Fourteen neurologically normal elderly (mean 68 ± 6 years) and 12 young (mean 27 ± 3 years) subjects participated. Under two postural conditions, upright standing (STD) and sitting (SIT), stretch reflex electromyographic (EMG) responses in the SOL/TA muscle were elicited by imposing rapid ankle dorsi-/plantar-flexion. Under the SIT condition, subjects were asked to keep the SOL background EMG level, which is identical to that under the STD condition. In the SOL muscle, both groups showed significant enhancement of the short-latency stretch reflex (SLR) response when the posture changed from SIT to STD. In the TA muscle, the young group showed significant enhancement of the middle- (MLR) and long-latency stretch reflex (LLR) when the posture changed from SIT to STD; no such modulation was observed in the elderly group. Since the TA stretch reflex responses under the STD condition were comparable in the young and elderly groups, the lack of posture-related modulation of the TA muscle in the elderly group might be explained by augmented stretch reflex excitability under the SIT condition. The present results suggest that the (1) SOL SLR responses are modulated both in the young and elderly subjects when the posture is changed from SIT to STD, (2) TA MLR and LLR responses are not modulated in the elderly subjects when the posture is changed from SIT to STD, while each response is same between the young and elderly in STD, and (3) the effect of aging on the posture-related stretch reflex differs in the SOL and TA muscles.

Janus Kinase-2 Inhibition Induces Durable Tolerance to Alloantigen by Dendritic Cell-Stimulated Human T Cells, Yet Preserves Immunity to Recall Antigen

Abstract 1903Janus kinase 2 (JAK2) conveys signals from receptor binding by several inflammatory cytokines, including IL-6, IL-12, and IL-23, via phosphorylation of signal transducer and activator of transcription 3 (STAT3). These cytokines promote the development and expansion of T helper 1 (Th1) cells, which use IL-12, and Th17 cells, which use IL-6 and IL-23. Th1 and Th17 cells can in turn induce alloreactive end organ damage in GvHD. JAK2 is therefore a principle gatekeeper of alloreactivity and inflammation, and it represents an attractive target to control GvHD. We demonstrate that TG101348, a synthetic small molecule, highly selective JAK2 inhibitor, when present during initial and secondary encounters between human T cells and allogeneic monocyte-derived dendritic cells (moDCs), induces durable, profound, and specific T cell tolerance on reexposure to the same alloantigens. TG101348 ablates IL-6/JAK2-mediated phosphorylation of STAT3 in T cells (24% v 0%, P<0.05, n=5) but has no off-target effects on IL-2 or IL-15/JAK3/pSTAT5-dependent signaling, which would interfere with Tregs and other effector T cell responses. JAK2 inhibition preserves Treg numbers and thereby enhances the ratio of CD4+ Tregs to CD8+CD25+ effector T cells in favor of Tregs (Treg:Effector 1:2 vs 1:1, P<0.05, n=5). JAK2 inhibition also reduces the production of IL-6 (255 vs 157 pg/ml, P<0.05, n=5) and TNF-alpha (50 vs 15 pg/ml, P<0.05, n=4) in allogeneic MLRs, impairing the activation of central and effector memory T cells as well as the expansion of responder Th1 (24% vs 14%, P<0.05, n=4) and Th17 (2% vs 1%, P<0.05, n=4). T cells, stimulated by moDCs in the presence of TG101348, demonstrate a marked reduction in proliferative capacity upon reexposure to fresh allogeneic moDCs from the original stimulating party, even in the absence of the JAK2 inhibitor during the secondary MLR (P<0.05, n=4). Responses to stimulation de novo by influenza matrix peptide (fluMP), a pathogenic nominal antigen, remain intact (P=NS, n=4). Conversely, TG101348 also inhibits secondary MLR responses by T cells, which have already been sensitized to alloantigens in a primary MLR without the JAK2 inhibitor (P<0.05, n=4). The durable tolerance against full HLA disparities in each of these settings provides preclinical data in vitro that JAK2 represents a relevant biologic target for preventing or treating graft-versus-host disease (GvHD), or even solid organ allograft rejection, without broader immune impairment. Disclosures:No relevant conflicts of interest to declare.

Intrathymic cortisone-resistant memory cells (46.11)

Abstract It is known that central memory cells CD8+ are found in immunocompetent lymphoid organs, such as spleen and lymph nodes. Previously, we described the approach allowing to see and estimate selective proliferation of memory cells in MLR with heat-silenced (45 degrees, 1 h) allogeneic stimulator splenocytes. We immunized C57BL/6 (H-2b) mice intraperitoneally with mastocytoma P815 cells (H-2d). Two months later, we tested the proliferation of thymocytes in the MLR with heat-silenced syngeneic C57BL/6 (H-2b), allogeneic B10.D2 (R101) (KdIdDb) and third-party FVB (H-2q) splenocytes. Thymocytes of intact mice did not respond in MLR at all. Thymocytes of immune mice displayed small, but significant MLR responses to heat-silenced allogeneic splenocytes, but not syngeneic or third-party splenocytes. To test the hypothesis that thymic memory cells are cortisone-resistant, we treated immune mice with hydrocortisone. Injection of high dose of hydrocortisone (2,5 mg) 2 days before isolation of responder thymocytes resulted in highly elevated MLR responses. This response was antigen-specific because “immune” thymocytes responded only on heat-silenced R101, but not syngeneic or third-party splenocytes. As expected, in proliferating cultures, the ratio of CD4/CD8 was less than 1 due to proliferation of memory CD8+ cells, responding on H-2Kd MHC class I molecule represented on R101 thymocytes. Thus, we have found that the thymus contained memory-like T cells, which are cortisone resistant.

B Cells as a Therapeutic Target for IFN-β in Relapsing–Remitting Multiple Sclerosis

IFN-β-1b is a first-line immunomodulatory therapy for relapsing-remitting multiple sclerosis (RR MS). However, its effects on B cells have not been characterized. In vitro studies of B cells derived from RR MS patients revealed that IFN-β-1b decreases B cells' stimulatory capacity, as detected by inhibition of the Ag-specific T cell proliferative response upon Ag presentation by IFN-β-1b-treated B cells. Our study has identified that IFN-β-1b inhibited B cells' stimulatory capacity in RR MS patients and healthy controls through the suppression of CD40 and CD80 expression, whereas the MHC class I and II expression was not changed. IFN-β-1b in vitro treatment inhibited B cell secretion of IL-1β and IL-23 and induced IL-12 and IL-27. Supernatants transferred from IFN-β-1b-treated B cells inhibited Th17 cell differentiation, as they suppressed gene expression of the retinoic acid-related orphan nuclear hormone receptor C and IL-17A and secretion of IL-17A. In addition, IFN-β-1b induced B cells' IL-10 secretion, which may mediate their regulatory effect. Studies of B cells derived from RR MS patients treated with recombinant s.c. injected IFN-β-1b revealed that they induced a significantly lower proliferative response in allogenic MLR than the B cells from untreated patients. Further confirming the IFN-β-1b in vitro-induced changes in B cell cytokine secretion, B cells derived from the IFN-β-1b-treated patients secreted significantly lower levels of IL-1β and IL-23 and higher levels of IL-12 and IL-27 in comparison with the B cells derived from untreated patients. We conclude that IFN-β-1b exerts its therapeutic effects in part by targeting B cells' functions that contribute to the autoimmune pathogenesis of RR MS.

In Vitro Assessment of Tolerance and Rejection Occurring After Co-Transplantation of Allogeneic Umbilical Cord Blood and Haploidentical CD34+ Cells as Treatment for Severe Aplastic Anemia

AbstractAbstract 2352 Introduction/Methods:The administration of highly purified haploidentical peripheral blood CD34+ cells combined with an unrelated cord blood transplant results in earlier neutrophil engraftment than is typically seen with a cord blood transplant alone. Chimerism data from pilot trials evaluating this strategy have reported 3 phases of engraftment: 1) early myeloid engraftment from transplanted haplo-CD34+ cells followed by 2) cord blood engraftment resulting in dual chimerism and 3) the subsequent disappearance of haploidentical donor cells with resultant full donor cord chimerism. The mechanism accounting for the disappearance of haploidentical cells has not been defined. Here the clinical results and an in vitro assessment of alloreactivity in three patients that underwent combined haploidentical CD34+ cell and cord blood transplantation for severe aplastic anemia (SAA) are described. The conditioning regimen consisted of cyclophosphamide (60mg/kg/day on days -7 and -6), fludarabine (25mg/m2/day on days -5 to -1), horse ATG (40mg/kg/day on days -5 to -2), and total body irradiation (200cGy on day -1). GVHD prophylaxis consisted of tacrolimus and mycophenolate mofetil. PCR of STRs was used to assess chimerism in T-cell and myeloid lineages and mixed lymphocyte reaction assays(MLR) were performed on peripheral blood samples collected at different time-points post-transplant to assess for alloreactivity against the recipient, the haploidentical donor, or the cord unit. Stimulator cord blood cells for the MLR were obtained from residual cord blood cells remaining in the infusion bag after patient administration and expanded in vitro using anti-CD28/CD3 Dynabeads. Results:Prior to transplantation, all three pts had transfusion dependent SAA associated with severe neutropenia that was refractory to conventional immunosuppressive therapy. Pt 1 had an early transient myeloid recovery (ANC 400 on day+11) from the haploidentical donor followed by engraftment of the cord unit (Cord ANC > 500) on day 21. The patient is currently 2 years post transplant and has 100% cord blood chimerism and is transfusion independent. An MLR assay performed when donor T-cell chimerism was 100% cord, showed evidence for rejection of the haploid cells by cord blood T-cells, with the MLR response to haploidentical donor cells being seven fold higher than the response to fully HLA-mismatched 3rd party cells. In pt 2, neutrophil recovery from the transplanted haploidentical donor occurred on day +10, with chimerism studies showing no evidence for cord engraftment in either myeloid or T-cell lineages at any point post-transplant. The patient is currently 15 months post transplant and is transfusion independent with normal blood counts and sustained “split” chimerism (T-cells recipient in origin with myeloid cells being 100% haploidentical donor). MLR assays showed that the recipient was tolerant to the haploid donor, with no statistically significant difference in the alloreactive response to the haploid donor compared to self. In pt 3, neutrophil recovery from the transplanted haploidentical donor occurred on day +10, with chimerism studies showing split chimerism (T-cell chimerism >90% cord and myeloid chimerism 88–100% haploid donor in origin). MLR assays again showed evidence of rejection of the haploid cells by cord blood T-cells, with a trend towards greater alloreactivity against the haploid donor compared to an HLA mismatched 3rd party on post-transplant day +63. Conclusions:Combined haploidentical CD34+ cell and unrelated cord blood transplantation following highly immunosuppressive conditioning represents a viable treatment option for patients with SAA who lack an HLA-matched donor. Using this approach, 2 of 3 pts had cord blood engraftment associated with early neutrophil recovery from the haploidentical donor. In one pt, the cord unit failed to engraft. Remarkably, sustained engraftment from the haploidentical donor in this pt resulted in transfusion independence. MLR appears to be a useful approach to assess the in vitro alloreactivity of this unique stem cell graft source. In the two pts who had cord engraftment, in vitro MLR assessments established that the disappearance of haploid cells occurred as a consequence of rejection of the haploidentical cells by engrafting cord blood T-cells, rather than from non-immunological haploidentical cell graft failure. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Immune “tolerance profiles” in donor bone marrow infused kidney transplant patients using multiple ex vivo functional assays

Ex vivo identification of donor-specific unresponsiveness in organ transplant recipients is important for immunosuppression (IS) minimization. We tested three groups of stable living, related-donor kidney transplant patients up to 11 years postoperatively, i.e., 20 haploidenticals with donor bone marrow cell (DBMC) infusions, eight noninfused haploidentical controls (haplo controls), and 11 HLA-identical controls (HLA-id), using multiple ex vivo immune assays. We observed that no patients developed donor-specific antibodies. The majority showed donor-specific CTL unresponsiveness from year 1 onward. Thirteen of 20 DBMC recipients became specifically donor MLR nonreactive. Depletion of donor cells in DBMC recipients still MLR reactive increased donor-specific reactivity by 75% ± 36% ( p = 0.04). Adding them back in low concentration caused antigen specific inhibition. The frequencies of ELISPOT granzyme-B and interferon-γ–producing cells somewhat paralleled the CTL and MLR responses. In the trans vivo DTH, 14 of 19 DBMC recipients demonstrated donor-specific unresponsiveness and 16 of 19 showed “linked suppression,” vs none of eight and one of eight haplo controls and vs six of 10 and one of 10 HLA-ids, respectively. Most importantly, when all six assays were performed simultaneously, 10 of 18 DBMC, five of 10 HLA-ids, and no haplo controls were specifically donor unresponsive long term. We propose that a cluster analysis combining these assays will reveal tolerant recipients in whom IS minimization may safely be tested. This appears to have occurred in many DBMC-infused recipients.

Facilitating Engraftment After MHC-Matched, Allogeneic BMT by IL-2 / Anti IL-2 Complex Treatment Requires Targeting CD25 On, and Activation in Situ of, Residual CD4 Tregs.

Abstract 66We recently demonstrated that administration of IL-2 complexed to the anti IL-2 antibody JES6-A12 (IAC) induced stable chimerism and engraftment of donor HSCT (BBMT 15:785, 2009). Based on suppression of anti-donor MiHA-specific host T cells, it was concluded that IAC administration enhanced chimerism by suppression of HVG. We proposed that in situ manipulation of host Tregs was crucial to facilitating engraftment and establishing tolerance in this model and hypothesized that the enhanced chimerism induced by this strategy was a direct result of host Treg activation, expansion and function following engagement of the IL-2 receptor CD25. To directly test this hypothesis, B6 CD4−/− (H-2b, Ly9.1−) mice were infused with highly enriched CD4 cells from B6-WT or B6-IL-2Rβ−/− mice deficient in CD25 expression and 4 days later conditioned with 5.5 Gy TBI. One day later, these mice were transplanted with MHC-matched, MiHA-mismatched 4 × 106 BALB.B (H-2b, Ly9.1+) TCD-BM. At days +3 and +5, all recipients were administered IAC and subsequently assessed for peripheral donor chimerism. By 2 weeks post-HCT, untreated control mice had increased circulating levels of CD8TETRAMER+ T cells (representing specific host anti-donor H60 MiHA reactive T cells) vs. IAC-treated recipients. Three months post-HCT, CD4−/− recipients of WT but not IL-2Rβ−/− CD4 cells were chimeric as evidenced by high levels of circulating donor cells (60% vs. <1%). These findings demonstrate that IAC effects require host CD25+ Treg cells and we propose that facilitation of engraftment by this strategy was a direct result of Treg cell activation and expansion following engagement of IAC with CD25. To assess Treg activation, we examined these cells in our standard BALB.B à B6 HSCT model. Tregs isolated 7 days post-HCT from IAC-treated but not untreated recipients expressed readily demonstrable levels of pStat-5a expression (∼2X increase in IAC-treated vs. PBS controls). Moreover, culture of the former Tregs in the presence of rmIL-2 illustrated their heightened sensitivity to activation by this cytokine as virtually all Tregs from IAC-treated animals exhibited high levels of pStat-5a expression (3.2 × 106 FoxP3+ P-Stat5a+ cells ± SE 0.6) compared to control mice (0.4 × 106 FoxP3+ P-Stat5a+ cells ± SE 0.5). Following this activation, increased numbers of host CD4+ FoxP3+ Tregs were readily identified in the PB and spleen of IAC vs. PBS-treated recipients. We next directly examined functional capacity of residual host Tregs exposed to IAC post-5.5Gy TBI and-transplant. Eight days post-HSCT, host Tregs (0 – 25 × 103/well) were isolated and highly enriched populations assessed for suppression of TCONV in anti-CD3 activation assays. Tregs from IAC-treated recipients efficiently mediated suppression at least equivalent to that by normal, untreated Tregs in these assays. Additionally, these IAC treated residual B6 Tregs also effectively inhibited allogeneic MLR responses by B6 CD4+ CD25− responder cells. Therefore, the capacity of host Tregs to respond to IAC activation signals and suppress T cell activation remained intact following conditioning and HSCT. We conclude that host Treg cell activation/expansion is central to the suppression of host effector cell responses to donor hematopoietic antigens leading to the inhibition of HVG following IAC administration resulting in enhanced engraftment. Disclosures:No relevant conflicts of interest to declare.

A Novel Therapy for Autoimmune Arthritis Using CD40 Gene‐Silenced Dendritic Cells

CD40 plays a critical role in dendritic cell (DC)‐mediated immune modulation. We have previously achieved immune modulation through RNA interference (RNAi), implying the therapeutic potential of RNAi‐modified DC as antigen‐specific tolerogenic therapies. To date, an RNAi‐based treatment for autoimmune arthritis is not reported. We hypothesized that treatment with CD40‐silenced DC may develop antigen‐specific tolerance and may be beneficial to the prevention and intervention of autoimmune arthritis. To test this, we generated DC and knocked down CD40 using small interfering RNA (CD40‐siRNA). CD 40 silenced DC displayed inhibitory capacity of T cell response in MLR. Immunization with type II collagen (CII)‐pulsed and CD40 gene‐silenced DC (CII‐pulsed/gene‐silenced DC) resulted in antigen‐specific nonresponsiveness in T cell responses. Vaccination with CII‐pulsed/gene‐silenced DC prevented collagen‐induced arthritis (CIA) disease development in a murine rheumatoid arthritis model. Furthermore, administration of CII‐pulsed/gene‐silenced DC at pre‐clinic stage was sufficient to inhibit progression of CIA. The therapeutic effects were further evidenced by decreased clinical scores, inhibited inflammatory infiltrates, and suppressed T cell and B cell responses to CII. In conclusion, this study is the first demonstration of immunotherapy using CD40 silenced DCs for autoimmune arthritis.

A Role for Regulatory Cells in Tolerance Induction in Recipients of Haploidentical Combined Non-Myeloablative Bone Marrow and Kidney Transplantation?.

Following an in vivo T cell depleting non-myeloablative conditioning regimen, 5 patients, aged 22–49, received combined kidney and bone marrow transplantation from a haploidentical related donor. Rituximab was included in the conditioning for patients 4 and 5. All patients developed initial mixed chimerism but lost it by day 21; no patient developed GVHD. Four patients discontinued immunosuppression from 240 to 422 days after BMT and have remained off immunosuppression for 9 to 52 months with no evidence of allograft rejection. Flow cytometry was used to assess lymphocyte subsets recovering after transplant. CD3 counts recovered slowly, exceeding 500 cells/μl at days +271, +365, +640 and +450. While memory CD45RO+ cells were most prevalent among CD4+ cells, naïve-type CD4+CD45RA+ cells, presumably arising from the recipient thymus, ranged from 8% to 56% at the time when total CD4 counts recovered to >100 cells/μl (days +165, +21, +352, +240). Notably, a very high proportion of initially recovering T cells were CD3+CD4+ expressing CD25 in all patients as early as day 7 and persisted over 1 year in 2 patients. At approximately day +120 and +365, we further characterized these cells for CD127, FOXP3, CD45RO, CD45RA, HLA-DR and CD62L expression. At Day +120, all 4 patients showed increased frequencies (10.7±4.6%) of CD25+CD127-FOXP3+ regulatory T cells (Treg) within the CD4 population compared to healthy subjects (3.8±0.4%). Expression of CD45RO, CD45RA, CD62L and HLA-DR was variable. By 1 year post-transplant, frequencies of Treg had decreased to levels similar to those in normal subjects. In vitro assays for CD8 and CD4 T cell-mediated alloreactivity (CML/MLR) showed development of long-lasting donor-specific unresponsiveness by 3 months after transplant in Patients 2, 4 and 5, and by 9 months in Patient 1. Responses to 3rd party recovered in all patients after a period of unresponsiveness. In Patient 1, in whom anti-donor CML reactivity declined gradually to become unresponsive by 9 months, depletion of CD4+CD25+ cells revealed a residual anti-donor CML and MLR response at 1year but not at 18 months. In 2 other patients, depletion of CD4+CD25+ cells did not reveal an anti-donor response at time points analyzed from day +122 to 2 years. In patients in whom renal tubular epithelial cells (RTEC) were cultured from the donor kidney, loss of killing activity against donor RTEC was observed post-transplant. The high percentage of Treg recovering early after transplant suggests that they may play a role in initial tolerance induction. This regulatory mechanism may be followed by later deletion of donor-reactive T cells. The variable ability to detect regulation of anti-donor reactivity may reflect the strength of the initial response, as patients with weak pre-transplant anti-donor responses and rapid post-transplant development of donor unresponsiveness did not reveal anti-donor response when Treg were depleted. In addition, infiltration of Treg at the graft site, not revealed by the assays described, might be responsible for tolerance in these patients.

Donor Specific Unresponsiveness Develops Following Loss of Chimerism in Recipients of Haploidentical Combined Bone Marrow/Kidney Transplantation but Not in Recipients of Bone Marrow Transplant Alone.

Pre-clinical data in monkeys receiving non-myeloablative conditioning followed by MHC-mismatched kidney and bone marrow transplantation show that transient chimerism is sufficient to permit achievement of long-term tolerance to a simultaneous donor renal allograft. Our group has recently reported successful induction of tolerance to donor kidneys in patients with advanced multiple myeloma and renal failure through combined bone marrow and kidney transplantation in the HLA-identical setting. On the basis of these results, five end-stage renal failure patients without malignant disease received simultaneous kidney and bone marrow transplantation from haploidentical HLA mismatched related donor after non-myeloablative conditioning with MEDI-507 (anti-CD2 humanized mAb; MedImmune), cyclophosphamide, thymic irradiation and peritransplant cyclosporine. Transplantation of kidney and bone marrow were both performed on Day 0. All patients developed initial mixed chimerism but lost their chimerism by Day 21. We have analyzed three patients who successfully discontinued immunosuppression on Days +240, +422 and +244. At a follow-up of 47, 38, and 17 months, all three patients are off immunosuppression without allograft rejection. T-cell counts exceeded 100 cells/μL by Day +128, +21, +21, respectively, and a high early prevalence of CD4+CD25high cells was detected. Post-transplant in vitro alloreactivity assays (bulk MLR/CML) showed the development of long-lasting donor-specific unresponsiveness in all three patients. In patients in whom renal tubular epithelial cells (RTEC) were cultured from the donor kidney, no killing of donor RTEC was detected post-transplant. We also assessed alloresponses in chemorefractory lymphoma patients receiving haploidentical bone marrow transplantation with a similar conditioning regimen. In contrast to the recipients of combined kidney/bone marrow transplants, these patients showed sustained global hyporesponsiveness in CML and MLR. However, loss of donor chimerism was associated with the eventual appearance of measurable anti-donor CML and/or MLR responses. In contrast, donor-specific and host-specific unresponsiveness with strong anti-3rd party responses developed in a sustained mixed chimera who received haploidentical stem cell transplantation with a modification of this conditioning protocol (i.e. different dose of MEDI-507, Isolex-selected CD34+ cells from G-CSF mobilized PBMC and the addition of fludarabine).In summary, we have obtained proof of principle that durable multilineage mixed chimerism with donor- and host-specific tolerance can be achieved without GVHD in humans receiving haploidentical HCT. In recipients of combined kidney/bone marrow transplants but not in recipients of bone marrow alone, patients who lose chimerism develop donor-specific unresponsiveness. These studies point to a role for the renal allograft in maintaining long-term tolerance following loss of initial donor chimerism.

Natural killer cells prime the responsiveness of autologous CD4+ T cells to CTLA4‐Ig and interleukin‐10 mediated inhibition in an allogeneic dendritic cell–mixed lymphocyte reaction

Cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA4-Ig) and interleukin (IL)-10 are immunomodulatory molecules which target CD28 costimulation by acting either directly or indirectly on the CD80/86 receptors on dendritic cells (DCs). This study examined the effect of combined treatment with CTLA4-Ig and IL-10 on T-cell responsiveness in a dendritic cell-mixed lymphocyte reaction (DC-MLR). T cells derived from nylon wool enrichment (NWT cells) demonstrated 15% (P = 0.006) and 10% (P = 0.0015) inhibition of proliferation with suboptimal doses of IL-10 (5 ng/ml) and CTLA4-Ig (20 ng/ml), respectively. Combined treatment with both agents resulted in 38% inhibition (P = 0.004) of the MLR response compared with untreated controls. In contrast to NWT cells, which consisted of CD4+, CD8+ and CD56+ (NK) cells, purified CD4+ T cells were less responsive to immunomodulation by CTLA4-Ig and IL-10. Repletion of the CD4+ T cells with NK cells restored IL-10 and CTLA4-Ig mediated immunomodulation, suggesting a role for NK cells in the regulation of DC-T-cell interactions. The specific effect of NK cells on DC activation was demonstrated by CD80 up-regulation on DCs in the absence of T cells. However, in the absence of DCs, NK cells augmented the proliferation of autologous CD4+ T cells stimulated by anti-CD3 monoclonal antibody (mAb), which was blocked by CTLA4-Ig. It is proposed that, in the MLR, immunomodulation by suboptimal CTLA4-Ig and IL-10 is influenced by cellular interactions of NK cells with DCs and T cells involving DC lysis and costimulation. Thus, NK cells prime both DCs and T cells to low doses of CTLA4-Ig and IL-10 during alloimmune responses, providing evidence for the potential interaction between innate and adaptive immunity.

Mouse model for analysis of non-MHC genes that influence allogeneic response: recombinant congenic strains of OcB/Dem series that carry identical H2 locus

AbstractAlloreactivity is the strongest known primary immune response. Its clinical manifestations are graft rejection, graft-versus-host disease and graft-versus-leukemia effect. The strongest stimulation by allogeneic cells is due to incompatibility at the major histocompatibility complex (MHC) genes. However, the non-MHC genes also participate in allogeneic response. Here we present a mouse model for study of the role of non-MHC genes in regulation of alloreactivity and show that they besides encoding antigens also regulate the responsiveness. Recombinant congenic strains (RCS) of O20/A (O20)-c-B10.O20/Dem (OcB/Dem) series have been derived from the parental strains O20 and B10.O20, which carry identical MHC haplotypes (H2pz) and therefore their differences in alloantigen response depend only on non-MHC genes. We have tested a MLR response by spleen cells of the strains O20, B10.O20, and 16 OcB/Dem strains through stimulation by cells from strains C57BL/10 (H2b), BALB/c (H2d), CBA (H2k), and DBA/1 (H2q) alloantigens. Proliferative response of O20, B10.O20 and OcB/Dem strains to these four alloantigens exhibited a similar but not completely identical pattern of reactivity. The responses to different alloantigens were highly correlated: C57BL/10-BALB/c r = 0.87, C57BL/10-CBA r = 0.84, C57BL/10-DBA/1 r = 0.83. Cluster analysis of the responses by O20, B10.O20, and OcB mice identified groups of strains with distinct patterns of response. This data shows that two main types of genes influence MLR: 1. structural genes for major and minor alloantigens and 2. genes regulating T-cell receptor signal transduction or mediating costimulatory signals by antigen-presenting cells.

Analysis of Anthrax and Plague Biowarfare Vaccine Interactions with Human Monocyte-Derived Dendritic Cells

The anti-biowarfare anthrax and plague vaccines require repeated dosing to achieve adequate protection. To test the hypothesis that this limited immunogenicity results from the nature of vaccine interactions with the host innate immune system, we investigated molecular and cellular interactions between vaccines, dendritic cells (DCs), and T cells and explored the potential for adjuvants (pertussis) to boost induction of host immunity. Human monocyte-derived DCs were matured in the presence of vaccines and analyzed for their ability to induce Th1/Th2 development from naive T cells, expression of cell surface maturation/costimulation molecules, and cytokine production. The vaccines showed different behavior patterns. Although the plague vaccine is equivalent to control maturation factors in maturation and stimulation of DCs and induces strong MLR and Th outgrowth, the anthrax vaccine is a poor inducer of DC maturation, as indicated by low levels of HLA-DR, CD86, and CD83 induction and minimal proinflammatory cytokine production. Interestingly, however, anthrax vaccine-treated DCs stimulate Th1 and Th2 outgrowth and a limited MLR response. There was no sustained negative modulatory effects of the anthrax vaccine on DCs, and its limited stimulatory effects could be overridden by coculture with pertussis. These results were supported by analysis of anthrax vaccine recall responses in subjects vaccinated using pertussis as an adjuvant, who demonstrate anthrax-specific effector T cell responses. These data show that the anthrax vaccine is a suboptimal DC stimulus that may in part explain the observation that it requires repeated administration in vivo and offer a rational basis for the use of complementary DC-maturing adjuvants in combined immunotherapy.