Lymphocyte Immunotherapy Research Articles

Improving the chance of successful implantation – part 2 – Circumventing immune rejection and the fetal semi-allograft

Purpose: To review possible mechanisms of how the fetal semi-allograft avoids immune rejection. Based on these mechanisms potential therapies to improve implantation by suppressing immune rejection are discussed. Materials and Methods: Studies supporting the importance of attaining T suppressor (sup) cells to the maternal fetal interface, while decreasing TH 17 cells along with causing a shift from cellular immunity to humoral immunity by causing a shift of influence from TH1 to TH2 cytokines is presented. Also discussed is the importance of suppressing the ability of natural killer cells to attack the fetal semi-allograft related to the secretion of an immuno-immunomodulatory protein known as the progesterone induced blocking factor (PIBF). Results: Progesterone supplementation in the luteal phase, possibly by causing the production especially of intracellular PIBF, may be the most important therapy to improve embryo implantation by suppressing immune rejection. Other potential therapies include human chorionic gonadotropin (hCG) supplementation and lymphocyte immunotherapy. Conclusions: Knowledge of the mechanism by which the fetal semi-allograft escapes immune surveillance should lead to more novel therapies to improve embryo implantation.

Histoprognostic Factors in Bladder Cancer: A Case Series of 156 Patients

Background: For a long time, phenomenon of defective immune-surveillance in cancer patients from pathological standpoint was not elucidated. I have reported 1985 that lowering seral cyclic AMP is biochemical reason of defective immunesurveillance from adoptive lymphocyte immunotherapy experiment among cancer patients. Recently, Honjo Tasku et al., reported why T cell don’t attack cancer cells by the covering of PD-L1 on its receptor of PD-1. This is also one of defective immune-surveillance from cellular side. I have recently surveyed the correlation between seral cyclic AMP concentration and immune activity in cancer patients. I have confirmed the biochemical reason of lowering cyclic AMP leads to seral defective immune-surveillance in cancer patient’s serum. Methods: I have surveyed seral cyclic AMP concentration and T cell number and NK cell among 40 cancer patients and preclinical cancer patients. Results: The averaged seral cyclic AMP has a positive tendency between lower immune activity and lower cyclic AMP in the serum when cancer is developing beforehand. Conclusion: As cyclic AMP is elevated after fasting therapy, adoptive lymphocyte immunotherapy become useful as immuno-potentiation even if in advanced cancer patients.

Low-dose lymphocyte immunotherapy rebalances the peripheral blood Th1/Th2/Treg paradigm in patients with unexplained recurrent miscarriage

BackgroundThe published results regarding lymphocytes immunotherapy for unexplained recurrent miscarriage (uRM) patients are conflicting due to different screening criteria and therapeutic protocols. The objective of the present study is to evaluate the effectiveness of immunotherapy using low-dose lymphocytes in patients with uRM and Th1/Th2/Treg paradigm disorders.MethodsSixty-four uRM patients who received low-dose lymphocytes immunotherapy served as the immunotherapy group, while the other 35 women who did not receive the treatment served as the control group. The proportions of peripheral blood Th1 cells, Th2 cells and Treg cells; and the concentration of TGF-β1 in serum were detected by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively, before and after the immunotherapy.ResultsThe proportion of Th1 cells was significantly decreased while the proportions of Th2 cells and Treg cells were significantly increased in immunotherapy group patients after treatment. In addition, the concentration of TGF-β1 in serum was significantly higher after immunotherapy than before. Forty-three uRM patients achieved pregnancy after receiving immunotherapy and 5 patients underwent miscarriages in the immunotherapy group (11.6%, 5/43), while 8 of the 23 pregnant patients experienced a miscarriage in the control group (34.8%, 8/23; p < 0.05).ConclusionsLow-dose lymphocyte immunotherapy is beneficial for restoring balance in the Th1/Th2/Treg paradigm and improving pregnancy outcome in uRM patients.Trial registrationNCT03081325. ClinicalTrials.gov. Retrospectively registered July 2015.

In Vivo MR Imaging of Glioma Recruitment of Adoptive T-Cells Labeled with NaGdF4 -TAT Nanoprobes.

Adoptive T lymphocyte immunotherapy is one of the most promising methods to treat residual lesions after glioma surgery. However, the fate of the adoptively transferred T-cells in vivo is unclear, hampering the understanding of this emerging therapy. Thus, it is highly desirable to develop noninvasive and quantitative in vivo tracking of these T-cells to glioma for better identification of the migratory fate and to provide objective evaluation of outcomes of adoptive T-cell immunotherapy targeting glioma. In this work, ultrasmall T1 MR-based nanoprobes, NaGdF4 -TAT, as molecular probes with high longitudinal relaxivity (8.93 mm-1 s-1 ) are designed. By means of HIV-1 transactivator (TAT) peptides, nearly 95% of the adoptive T-cells are labeled with the NaGdF4 -TAT nanoprobes without any measurable side effects on the labeled T-cells, which is remarkably superior to that of the control fluorescein isothiocyanate-NaGdF4 concerning labeling efficacy. Labeled adoptive T-cell clusters can be sensitively tracked in an orthotopic GL261-glioma model 24 h after intravenous infusion of 107 labeled T-cells by T1 -weighted MR imaging. Both in vitro and in vivo experiments show that the NaGdF4 -TAT nanoprobes labeling of T-cells may be a promising method to track adoptive T-cells to improve our understanding of the pathophysiology in adoptive immunotherapy for gliomas.

Lymphocyte immunotherapy in the treatment of recurrent miscarriage: systematic review and meta-analysis.

Recurrent miscarriage (RM) affects up to 2-3% of couples of reproductive age. There are several causes for this condition, including immunologic. The embryo is considered an allograft, subject to the rejection mechanisms of the maternal immune system. Immunotherapy involving immunization with lymphocytes is considered in cases of idiopathic RM. However, there is still no consensus regarding the efficacy and safety of this therapy. This systematic review and meta-analysis evaluated the data available in the literature regarding the efficacy and safety of the use of immunotherapy with lymphocytes in couples with history of RM. Searches in PubMed/Medline, SCOPUS, and Cochrane Library databases were conducted, using the following keywords: "recurrent miscarriage," "lymphocyte immunotherapy," and "meta-analysis." Statistical analyses were performed using Review Manager 5.3 (RevMan), version 5.3. Six published meta-analysis were retrieved; two found no improvements in the rate of live births after the use of immunization with lymphocytes in the treatment of RM, and four found a beneficial effect of the use of immunotherapy with lymphocytes in cases of RM, with significant improvements in the rate of live births. Data available in the literature supports the efficacy and safety of immunotherapy with lymphocytes in cases of RM without an identified cause.

Lymphocyte immunotherapy performed is pregnant gilts and sows did not affect preweaning mortality in the corresponding litters.

Summary This study was designed to test if lymphocyte immunotherapy (LI) of pregnant gilts and sows could improve preweaning mortality rates. In a commercial farm, Gilts (n = 61) first (n = 57) or second parity sows (n = 42) were selected and random assigned to treatment with saline (control group) or LI with lymphocytes obtained from multiparous sows, Boar, or growth-fattening pigs donors (alloimmunized groups). Treatments were performed at day 85 of pregnancy. Litter size and weight were evaluated by multifactor analysis of variance, whereas farrowing and preweaning mortality rates were analyzed by Mann-Whitney test. All traits evaluated at farrowing and at weaning did not significantly differ between groups (p &gt; 0.05); when data from all immunized groups were altogether compared with control group (p &gt; 0.5). Enteritis tended to be the most common cause of morbidity (p = 0.53). Mortality rate tended to increase in piglets from alloimmunized females (p = 0.12). The results suggest that LI of pregnant cross-bred gilts, first or second-parity sows did not affect preweaning mortality in piglets from subsequent lactation. Keywords: farrowing, litter size, litter weight, mortality, swine, weaning. Resumen El presente estudio se diseñó para evaluar si la terapia con linfocitos alogénicos (LI) aplicada en cerdas gestantes podía reducir las tasas de mortalidad en lactancia de sus respectivas camadas. En una granja porcícola comercial se seleccionaron cerdas gestantes (80-90 días) de primera (n = 61), segunda (n = 42) gestación, y se asignaron al azar al tratamiento con 1) linfocitos alogénicos (grupos inmunizados) obtenidos de cerdas gestantes, machos reproductores o cerdos de engorde; o 2) con solución salina (grupo control). Los datos del tamaño y el peso de la camada al nacer y al destete se registraron y evaluaron por análisis de varianza multifactorial mientras que la mortalidad al nacer y en la lactancia se evaluaron por prueba de Mann-Whitney. Ninguno de los parámetros evaluados de la camada al nacer y al destete, presentaron diferencias estadísticamente significativas entre los grupos aloinmunizados y el grupo control (p &gt; 0,05). La enteritis mostro tendencia a ser mayor causa de mortalidad en lactancia (p = 0,053). La mortalidad presento tendencia a ser mayor en los lechones de las cerdas aloinmunizadas (p = 0,12). Los resultados sugieren que la terapia con linfocitos alogénicos, administrada en cerdas gestante de primera, segunda o tercera gestación, no afecta la mortalidad predestete de la subsecuente camada.

Signal Transducer and Activator of Transcription (STAT) signaling b profile at the single cell level: Novel insights in the association of FOXP3+ T regulatory cells with recurrent spontaneous abortions before and after lymphocyte immunotherapy (LIT)

Signal Transducer and Activator of Transcription (STAT) signaling b profile at the single cell level: Novel insights in the association of FOXP3+ T regulatory cells with recurrent spontaneous abortions before and after lymphocyte immunotherapy (LIT)

Allogenic Lymphocyte Immunotherapy for Unexplained Recurrent Spontaneous Abortion: A Meta-Analysis.

Allogenic lymphocyte immunotherapy (LIT) as a treatment for unexplained recurrent spontaneous abortion (URSA) is still controversial due to the lack of enough controls to evaluate its effectiveness. Eighteen randomized, placebo-controlled trials with LIT for URSA were included in the meta-analysis. Live birth rates for each group were extracted, and the overall odds ratio (OR) for LIT was calculated. The success rate of treatment group was significantly higher (OR 3.74, 95% CI 3.07 ~ 4.57). LIT performed before and during pregnancy had dramatically improved the live birth rate in women with URSA (OR 4.67, 95% CI 3.70 ~ 5.90). The overall OR was 5.25 (95% CI 4.16 ~ 6.64), which supports a low dose of lymphocytes for treating URSA. Our results indicate that LIT provides a significantly beneficial effect over placebo for URSA. LIT given before and during pregnancy is superior to LIT given only before pregnancy, and the lower doses per treatment (less than 100 × 106 lymphocytes or 100 mL peripheral blood) achieved a better outcome.

Computational scoring system to predict HLA immunogenicity

BackgroundOur preliminary work has indicated that donor HLA immunogenicity in kidney and haemopoietic stem-cell transplantation might be predicted by assessment of their aminoacid sequence and physicochemical properties. We have now created a novel computational algorithm to quantify structural and surface electrostatic potential differences between donor and recipient HLA and applied it to predict alloantibody responses in a unique patient cohort. MethodsWe examined 141 patients undergoing treatment for infertility with lymphocyte immunotherapy. Patients were injected subcutaneously with partners' lymphocytes, and serum samples were collected before and after immunotherapy to assess sensitisation. After four-digit HLA typing and HLA structural modelling, donor–recipient HLA comparisons were performed to determine electrostatic mismatch score (EMS-3D) and assess its ability to predict development of donor-specific alloantibody and overall sensitisation to HLA after immunotherapy. Overall sensitisation was expressed as calculated reaction frequency (cRF), a measure that denotes incompatibility with a UK pool of 10 000 organ donors. FindingsDonor EMS-3D was a strong predictor of overall HLA class I and class II sensitisation risk (cRF >15%) (odds ratio [OR] 5·68 per unit increase, 95% CI 1·84–17·49; p=0·002) with a receiver operating characteristic area under curve score of 0·68 for cRF of more than 15% (p=0·0046) and 0·71 for cRF more than 85% (p<0·0001). Donor HLAs with the highest EMS-3D (fourth quartile) were associated with a higher risk of development of donor-specific alloantibody both for HLA class I (OR 2·90, 95% CI 1·62–5·23; p=0·031) and for HLA class II (16·07, 9·11–142·68; p<0·0001). Physicochemical differences between donor and recipient HLA-DQ were strong predictors of developing HLA-DQ donor-specific alloantibody (30·00, 6·33–87·73; p<0·0001). InterpretationOur study provides compelling evidence that donor HLA immunogenicity can be predicted by assessment of their structural and physicochemical disparities to recipient HLA. If confirmed in larger studies, our findings might enable better assessment of transplant immunological risk before transplantation and could inform future policies on allocation of deceased-donor kidney and haemopoietic stem-cell transplants to maximise the benefits of transplantation. FundingRoyal College of Surgeons of England Research Fellowship, Kiel University Starter Grant, Evelyn Trust, Academy of Medical Sciences.

Влияние лимфоцитотерапии на фибринолитическую активность крови и иммунные реакции у пациенток с репродуктивными потерями в анамнезе

Влияние лимфоцитотерапии на фибринолитическую активность крови и иммунные реакции у пациенток с репродуктивными потерями в анамнезе

Altered Th17/Treg Ratio in Recurrent Miscarriage after Treatment with Paternal Lymphocytes and Vitamin D3: a Double-Blind Placebo-Controlled Study.

Recurrent miscarriage (RM) affects 2-5% of pregnant women. Paternal lymphocyte immunotherapy is a common treatment for RM patients but the outcome has not been consistent. Therefore, combined therapy with other immunosuppressive drugs such as 1a, 25-dihydroxy-vitamin-D3 (vitamin D3) may improve the outcome. To investigate the effect of vitamin D3 on the balance of two essential T cells subsets, T helper (Th) 17 and T regulatory (Treg) cells, which contribute to the immune tolerance during pregnancy. The expression levels of CD4 and forkhead box protein 3 (FOXP3) in Treg cells, and the expression levels of CD4 and IL-17 in Th17 cells, were evaluated pre- and 3 months post-immunotherapy in RM patients treated with a combination of paternal lymphocytes and vitamin D3 compared with RM patients receiving lymphocyte immunotherapy alone. Vitamin D3 therapy decreased the frequency of Th17 cells in addition to reducing the Th17/Treg ratio in peripheral blood of RM patients compared with the control group (p<0.05). Considering that RM patients have a higher Th17/Treg ratio in peripheral blood, vitamin D3 may be a candidate therapeutic approach in this disease.

Anti-B lymphocyte immunotherapy is associated with improvement of periodontal status in subjects with rheumatoid arthritis.

Rheumatoid arthritis (RA) and periodontitis present many similar features. The benefits of anti-B lymphocyte therapy (rituximab) on reducing tissue resorption in RA prompted us to assess its potential efficacy on the periodontal status of patients with RA treated with rituximab. Periodontal status was assessed in 21 subjects with RA, divided into two groups: Group I consisted in 11 subjects assessed before their first infusion of rituximab and again 6 months later. Five of them were also assessed for up to 4 years after their first rituximab infusion. The 10 subjects in group II had received more than two courses of two rituximab infusions at the time of periodontal assessment. Pocket depth and attachment loss were significantly decreased 6 months after treatment with rituximab in group I. The periodontal status of the five subjects from group I followed for up to 48 months after rituximab treatment was improved irrespective of the clinical parameter observed. Patients from group II had a better periodontal status than patients from group I before treatment with rituximab. Anti-B lymphocyte therapy could be beneficial to improve periodontitis suggesting a major role of B cells in this disease.

The nitric oxide radical scavenger carboxy-PTIO reduces the immunosuppressive activity of myeloid-derived suppressor cells and potentiates the antitumor activity of adoptive cytotoxic T lymphocyte immunotherapy

Adoptive immunotherapy with cytotoxic T lymphocytes (CTLs) can result in robust and durable antitumor responses. Tumor-infiltrating CTLs produce IFNγ and mediate antitumor activity, but they simultaneously induce counter-regulatory immunosuppressive mechanisms in the tumor by recruiting monocytic myeloid-derived suppressor cells (MDSCs) that limit their proliferation and effector function. Using a murine model of adoptive immunotherapy for B16 melanoma, we developed a strategy to augment CTL activity by downregulating immunosuppression by MDSCs. Intravenous injection of transgenic pmel-1 CTLs into tumor-bearing mice, resulted in their infiltration into the tumor, but this was accompanied by the accumulation of large numbers of monocytic MDSCs (M-MDSCs). These cells hampered CTL function and reduced their numbers in the tumor. We determined that one mechanism responsible for this immunosuppression was the production of nitric oxide (NO) by MDSCs in the tumor. Therefore, mice were given the NO scavenger carboxy-PTIO (C-PTIO) on the day after CTL transfer. This led to the restoration of impaired proliferative capacity and function of the CTLs, resulting in sustained suppression of tumor growth. Thus, we conclude that CTL therapy can be improved by counter-acting immunosuppression. Targeting NO, one mediator of the immunosuppressive activity of M-MDSCs, may be an appropriate strategy to restore impaired CTL function and improve the efficacy of immunotherapy.

HCMV seroprevalence in couples under infertility treatment

Congenital HCMV infection affects about 0.5-0.9 % of newborns in Europe. Primary prevention could effectively be implemented in screening programmes for couples treated for infertility. By supplying epidemiological data, the proportion of women susceptible to primary HCMV infection is estimated. Retrospective evaluation of HCMV IgG of couples with recurrent implantation failure in German IVF programmes referred to a tertiary immunological centre for lymphocyte immunotherapy in 2002 (727 couples) and 2012 (294 male partners only). HCMV seroprevalence was 45 % in women and 33 % in men of childbearing age in 2002 and has not declined any further in the male group (34 %) in 2012. It was higher in women (39.8 %) than in males (28.0 %) of German descent (p < 0.00001). Most couples were concordantly seropositive (20.5 %) or negative (41.8 %). Discordant partnerships comprised of seropositive women with negative partners (24.6 %) and seronegative women with positive partners (13.1 %). More than 50 % of the women are HCMV seronegative and may benefit from primary prevention. About one fourth of them live with a seropositive partner. Risks to contract HCMV via the sexual route in a long-term discordant partnership may be very low, and the advice of protective safer sex methods in this group of prospective parents is critical since it leads to a conflict of interests.

Phase I trial of adoptively transferred tumor-infiltrating lymphocyte immunotherapy following concurrent chemoradiotherapy in patients with locoregionally advanced nasopharyngeal carcinoma

Adoptive cell therapy (ACT) for cancers using autologous tumor-infiltrating lymphocytes (TILs) can induce immune responses and antitumor activity in metastatic melanoma patients. Here, we aimed to assess the safety and antitumor activity of ACT using expanded TILs following concurrent chemoradiotherapy (CCRT) in patients with locoregionally advanced nasopharyngeal carcinoma (NPC). Twenty-three newly diagnosed, locoregionally advanced NPC patients were enrolled, of whom 20 received a single-dose of TIL infusion following CCRT. All treated patients were assessed for toxicity, survival and clinical and immunologic responses. Correlations between immunological responses and treatment effectiveness were further studied. Only mild adverse events (AEs), including Grade 3 neutropenia (1/23, 5%) consistent with immune-related causes, were observed. Nineteen of 20 patients exhibited an objective antitumor response, and 18 patients displayed disease-free survival longer than 12 mo after ACT. A measurable plasma Epstein–Barr virus (EBV) load was detected in 14 patients at diagnosis, but a measurable EBV load was not found in patients after one week of ACT, and the plasma EBV load remained undetectable in 17 patients at 6 mo after ACT. Expansion and persistence of T cells specific for EBV antigens in peripheral blood following TIL therapy were observed in 13 patients. The apparent positive correlation between tumor regression and the expansion of T cells specific for EBV was further investigated in four patients. This study shows that NPC patients can tolerate ACT with TILs following CCRT and that this treatment results in sustained antitumor activity and anti-EBV immune responses. A larger phase II trial is in progress.

Risk factors associated with a new pregnancy loss and perinatal outcomes in cases of recurrent miscarriage treated with lymphocyte immunotherapy

Objective: To assess the perinatal outcomes and risk factors for further pregnancy loss in patients with recurrent miscarriage treated with lymphocyte immunotherapy (LIT).Methods: We performed a retrospective observational study of women with a history of two or more consecutive miscarriages who underwent LIT. All patients had undergone investigation of the etiology of the pregnancy losses according to a specific protocol. These etiologic factors were compared between those whose pregnancy outcome was successful and those who had a further miscarriage. The comparison between the groups was performed by Kruskal–Wallis, Fisher exact and Chi-square tests. Perinatal outcome data were collected for the successful pregnancies.Results: One-hundred six patients were included. The mean number (±SD) of previous pregnancies, deliveries and miscarriages in all patients were 2.73 ± 0.8, 0.19 ± 0.4 and 2.54 ± 0.6, respectively. A successful pregnancy outcome after lymphocyte therapy occurred in 82 patients (group I), while 24 (22.6%) sustained a further miscarriage (group II). There was no statistical difference in the genetic, anatomic and hormonal causes of miscarriage between the groups (p > 0.05). Antinuclear (ANA) and antithyroglobulin (TgAb) autoantibodies occurred more frequently in group II (p = 0.0010 and p = 0.0024, respectively). Of those with successful pregnancies, 11 women (13.4%) had a preterm delivery. The mean birth weight was 3036.4 ± 498.6 g.Conclusion: In patients with recurrent miscarriage treated with LIT, the presence of ANA and TgAb was a risk factor for further pregnancy loss. Perinatal outcomes in those whose pregnancies continued were favorable.

Autoimmunity and thrombophilia in women treated with lymphocyte immunotherapy

able. Knowing that T cells infiltrate human decidua, we investigated the functional communications among DSC, dendritic cells (DC) and T cells, and particularly, the role of decidualized DSC on allogeneically triggered T cells by mature DC in mixed lymphocyte reaction (MLR). Methods: Decidual stromal cells from patients undergoing elective abortionswereharvested and expanded. The cellsweredecidualized in vitrowithmedroxyprogesterone, beta-estradiol and cAMP according to a standard protocol. These DSC were co-cultured with MLR, and proliferation and expression of CD25 (high affinity -chain of IL-2 receptor) and CD45RO (effector T cell isoform) on T cells were analyzed by FACS. Results: In previous reportswe showed thatDSC induce a slight proliferation of allogeneic T cells, which was even weaker when DSC were decidualized. Now, further assessment of the effect of the decidual microenvironment on effector T cells activated in MLR showed that when in a decidualized state, DSC suppress the proliferation rate of T cells to 70%. Furthermore, in contrast to CD4, cytotoxic CD8 T cells revealed a significant reduction inCD25andCD45RO markers suggesting amilder effector phenotype compared with MLR alone. Conclusion: Decidualized DSC can restrict proliferation and modulate the effector function of reactive T cells, and thus undoubtedly synergize with other physiological mechanisms for immune suppression. This study was supported by the projects: DOO250/2008 National Science Fund, Ministry of Education and Science; FP7-REGPOT-2009-1 ReProForce and BG051PO001-3.3.06-0059 the European Social Fund.

Lymphocyte Immunotherapy is Not Necessary for Primary Unexplained Abortions

Lymphocyte Immunotherapy is Not Necessary for Primary Unexplained Abortions

Characteristics of immune cell changes before and after immunotherapy and their clinical significance in patients with unexplained recurrent spontaneous abortion

To investigate the characteristics of immune cells before and after immunotherapy and their clinical significance in patients with unexplained recurrent spontaneous abortion (URSA), an analysis of 67 URSA patients, 67 sporadic spontaneous abortion (SA) patients, and 22 normal nonpregnant women (as controls) was conducted. URSA patients underwent immunotherapy using paternal lymphocytes. Peripheral blood from patients and controls was examined for lymphocytes and other markers of immune status. Before the immunotherapy, lymphocyte counts, CD4:CD8 cell ratios, and the relative proportion of natural killer (NK) cells were significantly higher in the URSA patient group than in the SA patient and control groups (P < 0.05). After the therapy, all of these three measures were decreased, whereas the percentage of T cells was increased, and statistically significant differences before and after the immunotherapy were detected (P < 0.05). Therefore, the immune system appears to be activated in the URSA patients, and the abnormal immunologic state in the URSA patients is more severe than in the SA patients. The alterations in T and NK cells may be involved in the etiopathogenesis of URSA. Lymphocyte immunotherapy appears to be an effective treatment for URSA patients.

Study on positive rate of blocking antibody in women with recurrent spontaneous abortion administered by route and frequency of paternal lymphocyte immunotherapy

To investigates factors affecting the positive rate of blocking antibody treated by paternal lymphocyte immunotherapy in patients with recurrent spontaneous abortion (RSA). From January 2008 to August 2012, 326 RSA cases undergoing treatment in Infertility Center of Qilu Hospital were studied retrospectively. Those patients were divided into 2 groups randomly: 260 cases in intradermal injection group were administered via bilateral forearm intradermal injections for immunotherapy once 21 days, then the blocking antibody was determined after 2 (23 cases) , 3(73 cases), 4 (74 cases) , 5(90 cases) times respectively, while in subcutaneous injection group, the 66 cases were administered via subcutaneous injection once 21 days, the blocking antibody measured after 3 times; In both cases, the blocking antibody was all determined 2 weeks later. The positive rate of blocking antibodies and the rate of successful pregnancy was recorded, and then followed up after the blocking antibody turning positive. (1) Positive rate of blocking antibodies:the positive rate of blocking antibodies were 17% (4/23) , 58% (42/73), 72% (53/74) and 84% (76/90) in the 2, 3, 4, and 5 times of intradermal injection group, respectively (P < 0.05). In subcutaneous injection group, the positive rate of blocking antibodies was 38 % (25/66), which was significantly lower than that in group intradermal injection receiving 3 times immunotherapy (P < 0.05). (2) The rate of pregnancy:the 176 patients out of 200 patients were pregnant when antibody was positive after immunotherapy, with 71.6% (126/176) of patients gained successful pregnancy(the length of pregnancy more than 5 months). The route and frequency of administration of immunotherapy could influence the positive rate of blocking antibody. The rate of successful pregnancy will be increased after blocking antibody turning positive.