Flt3 ligand expands lymphoid progenitors prior to recovery of thymopoiesis and accelerates T cell reconstitution after bone marrow transplantation

Deficient thymopoiesis and retarded recovery of newly developed CD4(+) T cells is one of the most important determinants of impaired immunocompetence after hemopoietic stem cell transplantation. Here we evaluated whether Fms-like tyrosine kinase 3 (Flt3) ligand (FL) alone or combined with IL-7 affects T cell recovery, thymopoiesis, and lymphoid progenitor expansion following bone marrow transplantation in immunodeficient mice. FL strongly accelerated and enhanced the recovery of peripheral T cells after transplantation of a low number of bone marrow cells. An additive effect on T cell recovery was not observed after coadministration of IL-7. Lineage(-)sca-1(+)c-kit(+)flt3(+) lymphoid progenitor cell numbers were significantly increased in bone marrow of FL-treated mice before recovery of thymopoiesis. Thymocyte differentiation was advanced to more mature stages after FL treatment. Improved T cell recovery resulted in better immunocompetence against a post-bone marrow transplantation murine CMV infection. Collectively, our data suggest that FL promotes T cell recovery by enhanced thymopoiesis and by expansion of lymphoid progenitors.     

Recovery of peripheral blood cells in irradiated mice pretreated with bacterial extract IRS-19

The effect of antigenic bacterial lysate IRS-19 on the recovery of blood cells was studied in mice injured by a single dose of 7 Gy irradiation. The preirradiation administration of IRS-19 accelerated the recovery of leukocytes, reticulocytes and platelets in peripheral blood. The recovery of leukocytes 9-14 days after irradiation in protected animals was accompanied by a higher level of band forms of granulocytes as well as activated lymphoid and monocytoid cells.     

B-lymphocyte differentiation in lethally irradiated and reconstituted mice. III. The influence of splenectomy on the recovery of the B-cell populations.

The recovery of the B-cell population was studied in irradiated and fetal liver-reconstituted mice. Since in irradiated and reconstituted mice the B-cell population in the spleen recovers much more rapidly than in the other lymphoid organs, we assessed the role of the spleen in the recovery of the B-cell compartment in the other organs. It was found that the absence of the spleen did not delay or diminish the recovery of the immunoglobulin (Ig)-bearing (B)-cell population in the bone marrow, lymph nodes, Peyer's patches, and peripheral blood. Throughout the recovery period the number of B lymphocytes in the lymphoid organs of splenectomized mice was even greater than in the same organs of sham-operated mice. B cells obtained from the bone marrow of splenectomized, irradiated, and reconstituted mice appeared to be fully immunocompetent, as shown by their ability to cooperate with thymocytes in an adoptive plaque-forming cell response to sheep red blood cells. The compensatory effect of the increased numbers of B cells in the bone marrow and peripheral lymphoid organs of splenectomized mice was reflected in the level of the serum immunoglobulins. Apart from a lower IgM concentration in the serum of splenectomized mice, no significant differences were found in IgG₁, IgG_2b, and IgA levels between splenectomized and sham-splenectomized mice. It is concluded that the spleen is not essential for both normal B-lymphocyte differentiation and maturation after irradiation and reconstitution.     

B-Lymphocyte differentiation in lethally irradiated and reconstituted mice. II. Recovery of humoral immune responsiveness.

The recovery of humoral immune responsiveness was studied in lethally irradiated, fetal liver-reconstituted mice. By means of both membrane fluorescence and antibody formation to sheep red blood cells (SRBC) as a functional assay, the rate of recovery of the compartments of B and T lymphocytes was determined in various lymphoid organs. The recovery of the immunoglobulin-positive (B) cell compartment after irradiation and reconstitution started in the spleen. This organ was also found to be the first in which the recovery of the B-cell population was completed. The interval between the recovery of the B-cell population in the spleen and that in the other organs tested was found to increase when the irradiated mice were reconstituted with spleen colony cells instead of fetal liver cells. This proved to be caused by the number and nature of the reconstituting hemopoietic stem cells. The immunoglobulin-positive (B) cells were found to appear before SRBC-reactive B cells could be demonstrated in spleen, lymph nodes, and Peyer's patches. The appearance of T lymphocytes in the various lymphoid organs required even more time. By means of cell transfer experiments, a sequential appearance of the precursors of anti-SRBC IgM-, IgG-, and IgA-plaque-forming cells could be demonstrated in spleen, bone marrow, lymph nodes, and Peyer's patches.     

The generation of large pyroninophilic cells in the lymphoid tissues of rats infused with cell-free lymph.

The thoracic duct of Wistar strain rats was cannulated during 5 days for studying the effect of selective lymphocyte depletion on the lymphoid tissue. A technique for the continuous infusion of cell-free lymph, whole lymph of Eagle's medium to the rat with the thoracic duct fistula is described in detail. The prolonged drainage of lymph from rats was followed by lymphopenia, sever atrophy of lymphoid tissues and the depletion of small lymphocytes in the thymus-dependent areas of spleen and lymph nodes. The infusion of cell-free lymph into the drained rat resulted in the recovery of the weight of lymphoid tissues and in the massive proliferation and accumulation of large cells with prominent nucleoli and intensely pyroninophilic cytoplasm in the lymphocyte depleted areas of the peripheral lymphoid tissues and thymic cortex. There was histological evidence that the large pyroninophilic cells developed well in the spleen and tended to localize preferentially around the periarteriolar region through the marginal zone bridging channels to the red pulp. The infusion of Eagle's medium was found ineffective in restoring the weight of the lymphoid tissues and in bringing about the proliferation of lymphoid cells. The rats infused with whole lymph showed almost similar findings biologically and histologically to those of sham-operated rats.     

Lymphocyte migration in syngeneic lymph node implants

Lymph nodes implanted subcutaneously to syngeneic recipients were shown to regenerate after mass cell destruction. Regenerated lymphoid tissue has a resemblance to the cortical zone of intact lymph nodes. Microenvironment of regenerated lymphoid tissue provides homing of lymphocytes. However, migration of 51Cr-labelled lymphocytes to implants declined drastically, as compared to lymphocyte migration to intact lymph nodes. Attenuation of proliferative activity and the data of morphological analysis indicate a more prolonged retention of lymphocytes in implanted lymph nodes. The results obtained could be attributable to only partial recovery of sinus and vessel systems regulating the inflow and outflow of lymphocytes in lymph nodes.     

Studies on recovery mechanism from rinderpest virus infection in rabbits. I. Effect of anti-thymocyte serum and thymectomy.

The role of cell-mediated immunity in recovery from rinderpest virus infection in rabbits was investigated by application of immunosuppressive procedures, i.e., treatment with anti-thymocyte serum and combined treatment with thymectomy and anti-thymocyte serum, both of which were confirmed to depress significantly cell-mediated immunity in rabbits. The immunosuppressed animals recovered in almost the normal fashion in terms of clinical signs, of virus clearance from the blood and lymphoid tissues and of repair of the lesions. It was suggested that the thymus-dependent cell-mediated immunity may not be essential in recovery from rinderpest virus infection. Possibility of participation of other recovery mechanisms was discussed.     

Stem cell factor consistently improves thymopoiesis after experimental transplantation of murine or human hematopoietic stem cells in immunodeficient mice

Deficient thymopoiesis is a pivotal determinant of impaired immune competence following hematopoietic stem cell transplantation (HSCT). Stem cell factor (SCF) is essentially involved in early thymopoiesis. We evaluated whether SCF administration would improve recovery of thymopoiesis following HSCT in immunodeficient mice receiving: 1) bone marrow (BM) transplantation of congenic mice; or 2) human fetal liver HSCT in the human immune system mouse model. Following murine BM transplantation, SCF significantly enhanced thymopoiesis and peripheral T cell recovery in lymph nodes and spleen. SCF did not affect BM lymphoid progenitor recovery and/or expansion. Median thymic cellularity increased from 0.9 in PBS- to 266 × 10(4)/thymus in SCF-treated mice (p = 0.05). Following human HSCT in human immune system mice, higher thymic cellularity was observed in SCF-treated mice. Double-negative and early double-positive thymocyte subsets increased, but especially late double-positive, CD4 single-positive, and CD8 single-positive thymocyte subsets were significantly enhanced (p < 0.05). These results show that exogenous supply of SCF may significantly improve murine and human posttransplant thymopoiesis, for which the effect is probably exerted by directly promoting T cell development intrathymically rather than by enhanced entry of prethymically expanded lymphoid progenitors.     

Effect of a single administration of testosterone on the immune response and lymphoid tissues in mice.

Female mice were given a single intraperitoneal injection of testosterone immediately after irradiation and marrow reconstitution. Thirty days later testosterone had no suppressive effect on the recovery of thymus and spleen weights. Testosterone had no effect on the graft-versus-host reaction. Testosterone had no influence on the survival of the skin homografts. However, the plaque-forming cell response to sheep erythrocytes in the spleen was dramatically suppressed by testosterone. Histological observations revealed marked inhibition of lymphoid regeneration selectively in the thymus-independent areas of the peripheral lymphoid tissues. These results suggest that testosterone would act mainly on the differentiation of stem cells toward the population of bone marrow-derived B lymphocytes. The immune response to sheep erythrocytes was restored completely 90 days after testosterone administration. Testosterone given to normal adult mice can also have suppressive activity on the immune system 30 days after a single intraperitoneal injection.     

Quantitative detection of hepadnavirus-infected lymphoid cells by in situ PCR combined with flow cytometry: implications for the study of occult virus persistence

The detection of small amounts of viral pathogens in infected cells by classical PCR is hampered by a partial loss of virus nucleic acid due to extraction and by difficulties in discrimination between truly intracellular virus genome material and that possibly adhered to the cell surface. These impediments limit reliable identification of virus traces within infected cells, which are typically encountered in latent and persistent occult infections. In this study, hepadnavirus-specific in situ PCR combined with the enzymatic elimination of extracellular virus and flow cytometry permitted detection of viral genomes in lymphoid cells without nucleic acid isolation and allowed quantification of infected cells during the course of persistent infection with woodchuck hepatitis virus (WHV). The validity of the procedure was confirmed by hybridization analysis of the in situ-amplified viral sequences. The results showed that hepadnavirus can be directly detected within lymphoid cells not only in serologically accountable infection, but also years after recovery from viral hepatitis and in the course of primary occult virus carriage. Percentages of infected peripheral lymphoid cells in symptomatic WHV hepatitis fluctuate between 3.4 and 20.4% (mean +/- standard error of the mean, 9.6% +/- 1.7%), whereas those in persistent, serologically mute WHV infection range from 1.1 to 14.6% (mean +/- standard error of the mean, 4.8% +/- 0.8%) (P = 0.005). The data obtained provide further evidence that WHV infection continues indefinitely in the lymphatic system independently of whether it is symptomatic or concealed. They document that hepadnavirus can be detected in a significant proportion of circulating lymphoid cells in both immunovirologically apparent as well as occult persistent infection.     

Natural history of woodchuck hepatitis virus infections during the course of experimental viral infection: molecular virologic features of the liver and lymphoid tissues.

In this study, the kinetic patterns of woodchuck hepatitis virus (WHV) infection were monitored in the liver and the five primary components of the lymphoid system (peripheral blood lymphocytes, lymph nodes, bone marrow, spleen, and thymus). Groups of woodchucks experimentally infected with a standardized inoculum of WHV were sacrificed at different times over a 65-week period beginning in the preacute phase of viral infection and continuing to the period of serologic recovery or the establishment of chronic infections and subsequent hepatocellular carcinoma. Infection by WHV was not limited to the liver but involved the major components of the lymphoid system during all stages of virus infection. A complex series of kinetic patterns was observed for the appearance of WHV DNA in the different lymphoid compartments and the liver during the entire course of viral infection. A progressive evolution of different WHV genomic forms related to the replicative state of WHV was also observed. Lymphoid cells of the bone marrow were the first cells in which WHV DNA was detected, followed in order by the liver, the spleen, peripheral blood lymphocytes, lymph nodes, and finally the thymus. Several differences were observed in the cellular WHV DNA patterns between woodchucks that developed chronic WHV infections and those that serologically recovered from acute WHV infections. The observations compiled in this study indicate that the host lymphoid system is intimately involved in the natural history of hepadnavirus infections from the earliest stages of virus entry.     

Cloning of murine B- and T-lymphocytes in vitro after the long-term action of tritium oxide

The cloning method was used to study the content of B- and T-lymphocyte committed precursors in central and peripheral organs of the immune system of mice at different times after long-term exposure to tritium oxide (a cumulative dose of approximately 9 Gy). It was shown that recovery of the colony-forming ability of the committed lymphocyte precursors was different in central and peripheral lymphoid organs; the dynamics and degree of restoration of the pools of B--(CFU--LB) and T--(CFU--LT) lymphocyte precursors were different.     

Improved method of ectopic transplantation of the whole spleen in mice

A simple and reliable technique has been developed for transplantation of the whole mouse spleen on the kidney. This technique ensures sufficient structural and functional recovery (transfer) of the original splenic microenvironments with a survival rate of 100%. The method allows counting the antibody-producing cells, as well as the spleen colony-forming units on the ectopic splenic territories. It may be used for studying hemopoietic and lymphoid microenvironments or for other special purposes.     

Antibody responses after Sendai virus infection and their role in upper and lower respiratory tract disease in rats.

BACKGROUND AND PURPOSE: Sendai virus infection in rats is an excellent model for studying development and role of host defenses throughout the respiratory tract after this infection. Therefore, development of serum antibody responses and disease were studied. METHODS: Forty-two anesthetized pathogen-free 3- to 4- week-old LEW/NCr rats were inoculated intranasally with Sendai virus. At postinoculation days 0, 2, 3, 5, 8, 10, and 14, rats were euthanized by administration of a pentobarbital sodium overdose followed by exsanguination. Serum was obtained from all animals, and nasal wash and bronchoalveolar lavage specimens were collected during selected experiments. An ELISPOT assay was used to measure numbers of Sendai virus-specific antibody-forming cells in respiratory tract lymphoid tissue. RESULTS: Recovery from disease and clearance of virus from respiratory tract tissues coincided with development of serum antibody responses. Upper respiratory tract lymph nodes were the initial and major sites of appearance of antibody-forming cells. Immunoglobulin G was the predominant subtype of these cells during recovery from the infection and in rats resistant to infection. Passive transfer of antisera or specific IgG protected the lower but not the upper respiratory tract. CONCLUSIONS: Circulating components of immunity have a major role in resistance and recovery from disease in the lower respiratory tract, whereas local responses are likely involved in protection of the upper respiratory tract. Local lymphoid tissues are the major production sites of IgG, which contributes to resistance to and recovery from respiratory tract diseases.     

Lesions and tissue distribution of viral antigen in severe acute versus subclinical acute infection with BVDV2.

Differences in the distribution and spread of viral antigen, development of lesions and correlation between presence of viral antigen and lesions were compared between a low and highly virulent strain of BVDV2. Two groups of two-week- to two-month-old colostrum-deprived calves were inoculated intranasally with the naturally occurring low virulent BVDV2 strain 28508-5 or the highly virulent strain 1373. To study the sequence of virus spread and lesion development, calves were necropsied at days three, six, eight-nine and 12 to 14 post inoculation (pi). Viral antigen was detected by the indirect immunoperoxidase method in cryostat sections and lesions were evaluated in H&E-stained paraffin sections. Clinical signs and changes in lymphocyte and thrombocyte numbers confirmed the difference in virulence between the two strains. Both strains showed comparable initial infection and spread at day three pi. At day six pi, they were found widespread in lymphoid tissues and multifocally in intestinal mucosa. Lesions were very mild despite the large amount of antigen in the lymphoid tissues. After day six pi, differences between the low and highly virulent strains became more prominent. The strain of low virulence was cleared from the tissues, but there was a transient phase of depletion. The highly virulent strain continued to spread to different organs and there was severe depletion of lymphoid tissues without recovery.     

孕期应激对子代大鼠骨髓淋巴干细胞发育的影响

孕期应激对子代产生的影响是多方面的,这种影响是复杂的。研究表明,出生前的应激经历可导致出生后子代长期的免疫功能改变。这些改变追其根源与骨髓淋巴干细胞的改变有关。本文综述了大鼠孕期经历应激的子代骨髓淋巴干细胞所受的影响及免疫系统的相关改变,并根据现有的研究提出假说,为进一步研究孕期应激导致子代免疫系统改变的机理研究提供新的思路。     

T and B cell recovery in arthritis adoptively transferred to SCID mice: antigen-specific activation is required for restoration of autopathogenic CD4+ Th1 cells in a syngeneic system

T cell homeostasis is a physiological function of the immune system that maintains a balance in the numbers and ratios of T cells at the periphery. A self-MHC/self-peptide ligand can induce weak (covert) signals via the TCR, thus providing an extended lifespan for naive T cells. A similar mechanism is responsible for the restoration of immune homeostasis in severe lymphopenic conditions such as those following irradiation or chemotherapy, or upon transfer of lymphocytes to nu/nu or SCID mice. To date, the genetic backgrounds of donor and recipient SCID mice were unmatched in all autoimmune arthritis transfer experiments, and the recovery of lymphoid cells in the host has not been followed. In this study, we present the adoptive transfer of proteoglycan (PG)-induced arthritis using unseparated and T or B cell-depleted lymphocytes from arthritic BALB/c donors to genetically matched syngeneic SCID recipient mice. We demonstrate that selectively recovered lymphoid subsets determine the clinical and immunological status of the recipient. We found that when T cells were depleted (>98% depleted), B cells did not produce PG-specific anti-mouse (auto) Abs unless SCID mice received a second Ag (PG) injection, which promoted the recovery of Ag-specific CD4(+) Th1 cells. Reciprocally, as a result of B cell recovery, high levels of serum anti-PG Abs were found in SCID mice that received B cell-depleted (>99% depleted) T lymphocytes. Our results indicate a selective and highly effective cooperation between CD4(+) T cells and B lymphocytes that is required for the restoration of pathological homeostasis and development of autoimmune arthritis in SCID mice.     

Analysis of time dependent changes of lymphopoietic activity in organ cultures of embryonic chicken thymus.

The time dependent development of lymphocytes in organ cultures of the thymus obtained from 10-day-old chick embryos was characterized by an initial phase of exponential increase of the number of lymphocytes per thymus followed by a plateau phase with no further increase in cell number. The proportion of cells in DNA synthesis dropped rapidly during the first 10 days of culture. Simultaneously the lymphocytes turned progressively smaller, as evidenced by both cell diameter and dry mass and constituted a homogeneous population of small cells at the end of the culture period. Thymic anlagen partially depleted of lymphoid precursor cells by a short hot pulse with 3H-TdR showed a prolonged exponential phase and reached normal plateau cell numbers 2-4 days later than usual. Furthermore, at least in the first part of the plateau phase, a reduction in the number of lymphoid cells per thymus resulted in a recovery in terms of the cell number which was associateed with increased DNA synthesis. These results are compatible with the regulation of thymic lymphopoiesis in organ culture through a mechanism operating via cell density.     

Late nonstochastic sequelae of gamma irradiation of lymphoid cells

In experiments with a suspension culture of human Raji lymphoid cells it was shown that a 40 per cent deceleration of the population growth induced by 2.5 Gy gamma-radiation persisted within the following 15-16 generations, afterwards it gradually normalized to reach the control level in the 21st generation. However, the incompleteness of recovery was displayed with the repeated exposure and cultivation of cells under hyperthermia (40 degrees C) up to the 27th generation.     

Dietary supplementation with probiotics improves hematopoiesis in malnourished mice

Background

Lactobacillus rhamnosus CRL1505 (Lr) administered during the repletion of immunocompromised-malnourished mice improves the resistance against intestinal and respiratory infections. This effect is associated with an increase in the number and functionality of immune cells, indicating that Lr could have some influence on myeloid and lymphoid cell production and maturation.

Objective

This study analyzed the extent of the damage caused by malnutrition on myeloid and lymphoid cell development in the spleen and bone marrow (BM). We also evaluated the impact of immunobiotics on the recovery of hematopoiesis affected in malnourished mice.

Methods

Protein malnourished mice were fed on a balanced conventional diet for 7 or 14 consecutive d with or without supplemental Lr or fermented goat''s milk (FGM). Malnourished mice and well-nourished mice were used as controls. Histological and flow cytometry studies were carried out in BM and spleen to study myeloid and lymphoid cells.

Results

Malnutrition induced quantitative alterations in spleen B and T cells; however, no alteration was observed in the ability of splenic B cells to produce immunoglobulins after challenge with LPS or CpG. The analysis of BM B cell subsets based on B220, CD24, IgM and IgD expression showed that malnutrition affected B cell development. In addition, BM myeloid cells decreased in malnourished mice. On the contrary, protein deprivation increased BM T cell number. These alterations were reverted with Lr or FGM repletion treatments since normal numbers of BM myeloid, T and B cells were observed in these groups.

Conclusions

Protein malnutrition significantly alters B cell development in BM. The treatment of malnourished mice with L. rhamnosus CRL1505 was able to induce a recovery of B cells that would explain its ability to increase immunity against infections. This work highlights the possibility of using immunobiotics to accelerate the recovery of lymphopoyesis in immunocompromised-malnourished hosts.