Chimerism, the Microenvironment and Control of Leukemia.

Transplantation of allogeneic hematopoietic cells faces two barriers: failure of engraftment due to a host versus graft reaction, and the attack of donor cells against the patient, the graft versus host (GVH) reaction. This reaction may lead to GVH disease (GVHD), but in patients transplanted due to leukemia or other malignant disorders, this may also convey the benefit of a graft versus leukemia (GVL) effect. The interplay of transplant conditioning with donor and host cells and the environment in the patient is complex. The microbiome, particularly in the intestinal tract, profoundly affects these interactions, directly and via soluble mediators, which also reach other host organs. The microenvironment is further altered by the modifying effect of malignant cells on marrow niches, favoring the propagation of the malignant cells. The development of stable mixed donor/host chimerism has the potential of GVHD prevention without necessarily increasing the risk of relapse. There has been remarkable progress with novel conditioning regimens and selective T-cell manipulation aimed at securing engraftment while preventing GVHD without ablating the GVL effect. Interventions to alter the microenvironment and change the composition of the microbiome and its metabolic products may modify graft/host interactions, thereby further reducing GVHD, while enhancing the GVL effect. The result should be improved transplant outcome.

On graphical tests for proportionality of hazards in two samples.

In this paper, we present a class of graphical tests of the proportional hazards hypothesis for two-sample censored survival data. The proposed tests are improvements over some existing tests based on asymptotic confidence bands of certain functions of the estimated cumulative hazard functions. The new methods are based on the comparison of unrestricted estimates of the said functions and their restricted versions under the hypothesis. They combine the rigour of analytical tests with the descriptive value of plots. Monte Carlo simulations suggest that the proposed asymptotic procedures have reasonable small sample properties. The power is much higher than existing graphical tests and comparable with existing analytical tests. The method is then illustrated through the analysis of a data set on bone marrow transplantation for Leukemia patients.

Δ9-Tetrahydrocannabinol attenuates allogeneic host-versus-graft response and delays skin graft rejection through activation of cannabinoid receptor 1 and induction of myeloid-derived suppressor cells.

Immune cells have been shown to express cannabinoid receptors and to produce endogenous ligands. Moreover, activation of cannabinoid receptors on immune cells has been shown to trigger potent immunosuppression. Despite such studies, the role of cannabinoids in transplantation, specifically to prevent allograft rejection, has not, to our knowledge, been investigated previously. In the current study, we tested the effect of THC on the suppression of HvGD as well as rejection of skin allografts. To this end, we studied HvGD by injecting H-2(k) splenocytes into H-2(b) mice and analyzing the immune response in the draining ingLNs. THC treatment significantly reduced T cell proliferation and activation in draining LNs of the recipient mice and decreased early stage rejection-indicator cytokines, including IL-2 and IFN-γ. THC treatment also increased the allogeneic skin graft survival. THC treatment in HvGD mice led to induction of MDSCs. Using MDSC depletion studies as well as adoptive transfer experiments, we found that THC-induced MDSCs were necessary for attenuation of HvGD. Additionally, using pharmacological inhibitors of CB1 and CB2 receptors and CB1 and CB2 knockout mice, we found that THC was working preferentially through CB1. Together, our research shows, for the first time to our knowledge, that targeting cannabinoid receptors may provide a novel treatment modality to attenuate HvGD and prevent allograft rejection.

Effect of isopropyl methylphosphonofluoridate (IMPF) poisoning on selected immunological parameters of angiogenesis.

INTRODUCTION AND OBJECTIVE: Acetylcholinesterase (AChE) and cholinergic receptors play an important role in the immune system, including lymphocyte-induced angiogenesis. However, their exact role is not fully understood. The presented work tests the influence of isopropyl methylphosphonofluoridate (IMPF), an irreversible inhibitor of AChE, on selected immune parameters associated with angiogenesis in mice. The levels of VEGF, bFGF, TNF-α, and IFN-γ production were measured, together with the ability of lymphoid spleen cells to induce local GvH reaction after a single dose of IMPF. MATERIALS AND METHOD: Experiments were performed in male BALB/c mice. Acetylcholinesterase activity in erythrocytes was determined by the Ellman`s procedure. Levels of cytokines were measured in serum using standard commercial ELISA kits. Influence of IMPF intoxication upon angiogenesis was examined by the LIA test, according to the Sidky and Auerbach procedure. RESULTS: The results showed a 6- and 8-fold increase in VEGF at days 1 and 7 of the experiment, respectively, as well as a decrease (at days 14 and 21 after administration), followed by a significant increase (day 1) in bFGF levels. A statistically significant decrease in the concentration of IFN-γ was observed throughout all experiments. The maximum decrease in the level of TNF-α was found at days 1 and 7 after administration of IMPF. Additionally, a significant decrease was found in the ability to form new blood vessels following IMPF administration. CONCLUSIONS: This study revealed that IMPF has a significant effect on the regulation of lymphocyte-induced angiogenesis, which is related with the modulation of angiogenic and pro-inflammatory cytokines secretion. The observed differences suggest a possible derangement of certain elements of the neuronal and/or non-neuronal cholinergic system.

Blockade of IL-6-signaling inhibits the pathogenesis of CD4+ T cell-mediated lethal graft-versus-host reaction against minor histocompatibility antigen.

Graft-versus-host reaction (GVHR) is considered as a problem in hematopoietic cell transplantation. We found that CD45RB(high) CD62L(+) naïve CD4(+) T cells from wild-type B10D2 (H-2d MMTV6(-)) mice immediately differentiated into effector T cells producing high-levels of various cytokines after the transfer into BALB/c RAG2(-/-) (H-2d MMTV6(+)) mice. The expanded CD4(+) T cells, which have almost TCR Vß3 chain, recognized the minor antigen of recipient mice and brought typical severe GVHR symptoms such as eyelid irritation, diarrhea, and liver failure. Eventually, all of the recipient mice transferred CD4(+) T cells was dead within 10 days. We demonstrated here that blockade of IL-6 signaling by administration of anti-IL-6 receptor (IL-6R) monoclonal antibody (mAb) remarkably inhibited the CD4(+) T cell-mediated lethal GVHR. In addition, we confirmed that the in vivo injection of anti-IL-6R mAb prevented the generation of effector CD4(+) T cells which produce the inflammatory cytokines such as IFN-γ, TNF-α, and IL-17. These findings indicated that IL-6 was a critical factor in the CD4(+) T cell-dependent acute GVHR induced by a minor-antigen, suggesting that IL-6-mediated signaling pathway would be a strong therapeutic target in T cell-mediated GVHR as well as other diseases including autoimmune and inflammation.

Limiting the amount and duration of antigen exposure during priming increases memory T cell requirement for costimulation during recall.

Donor-reactive memory T cells (Tmem) can play an important role in mediating graft rejection after transplantation. Transplant recipients acquire donor-reactive Tmem not only through prior sensitization with alloantigens but also through previous exposure to environmental pathogens that are cross-reactive with allogeneic peptide-MHC complexes. Current dogma suggests that most, if not all, Tmem responses are independent of the requirement for CD28 and/or CD154/CD40-mediated costimulation to mount a recall response. However, heterogeneity among Tmem is increasingly being appreciated, and one important factor known to impact the function and phenotype of Ag-specific T cell responses is the amount/duration of Ag exposure. Importantly, the impact of Ag exposure on development of costimulation independence is currently unknown. In this study, we interrogated the effect of decreased Ag amount/duration during priming on the ability of donor-reactive Tmem to mediate costimulation blockade-resistant rejection during a recall response after transplantation in a murine model. Recipients possessing donor-reactive Tmem responses that were generated under conditions of reduced Ag exposure exhibited similar frequencies of Ag-specific T cells at day 30 postinfection, but, strikingly, failed to mediate costimulation blockade-resistant rejection after challenge with an OVA-expressing skin graft. Thus, these data demonstrate the amount/duration of Ag exposure is a critical factor in determining Tmem's relative requirement for costimulation during the recall response after transplantation.

[Role of antithymocyte globulin in reducing the incidence of complications after allogeneic hemopoietic cell transplantation].

AIM: To evaluate the efficacy of antithymocyte globulin (ATG) used in conditioning modes before allogeneic hemopoietic cell transplantation (allo-HCT) and its effect in reducing the incidence of posttransplantation complications. SUBJECTS AND METHODS: The study assessed the results of 92 allo-HCTs depending on the presence or absence of ATG in conditioning modes, the doses of Atgam (60 mg/kg or more), the presence or absence of acute leukemia (AL) in remission before HCT. RESULTS: In patients with AL in remission receiving ATG in conditioning modes (Atgam 60 mg/kg or thymoglobulin 7.5 mg/kg), overall three-year survival was 60%. Increasing the dose of Atgam up to more than 60 mg/kg resulted in higher transplantation-associated mortality (TAM) rates than did with the Atgam dose of 60 mg/kg (p < 0.01). CONCLUSION: Allo-HCT is the treatment of choice for patients with AL in the presence of an HLA-identical related or unrelated donor. The use of Atgam in a course dose of not more than 60 mg/kg or thymoglobulin 7.5 mg/kg in conditioning modes is associated with low TAM rates and higher overall survival in earlier-stage disease in complete clinical hematological remission as compared with those in patients with expanded-stage AL, rather than in AL in remission at the start of conditioning before HCT.

Potassium humate inhibits carrageenan-induced paw oedema and a graft-versus-host reaction in rats.

It has been shown in a previous study that brown coal-derived potassium humate is safe and effective in suppressing contact hypersensitivity in rats. In this study the efficacy of potassium humate on other types of inflammation was determined. Preparative TLC followed by mass spectroscopy was used in an attempt to fingerprint the product. The effects of potassium humate, at an oral dose of 60 mg/kg bodyweight, on a delayed type hypersensitivity reaction, a carrageenan-induced inflammation model and an allogeneic graft-versus-host reaction (GVHR) in rats were investigated. Paw oedema was used as a measure of inflammation. It was found that potassium humate had no effect on the delayed type hypersensitivity reaction but significantly inhibited the increase in paw volume of the carrageenan-induced oedema in rats which compared favourably with indomethacin treatment. Furthermore, potassium humate inhibited the GVHR induced in normal and cyclophosphamide-treated immune-incompetent rats. The identification of a naturally occurring compound that is safe and effective in reducing different types of inflammation merits further evaluation in clinical trials.

T helper17 cells are sufficient but not necessary to induce acute graft-versus-host disease.

T helper (Th)1 cells were considered responsible for the induction of graft-versus-host disease (GVHD), but recently the concept has been challenged. Th17 cells play a critical role in mediating autoimmune diseases, but their role in the pathogenesis of GVHD remains unclear. Herein we compare the ability of in vitro generated Th1 and Th17 cells from C57BL/6 mice to induce GVHD in lethally irradiated BALB/c recipients. Allogeneic Th17 cells had superior expansion and infiltration capabilities in GVHD target organs, which correlated with their increased pathogenicity when compared with naïve or Th1 controls. Th17 cells caused no pathology in the syngeneic recipients, indicating that antigen-activation was required for their pathogenicity. Polarized Th17 cells could not maintain their phenotype in vivo as they produced a significant amount of interferon (IFN)-gamma after being transplanted into allogeneic recipients; however, IFN-gamma was not required for Th17 cell-induced GVHD. Further, we evaluated the pathogenesis of Th17 cells in GVHD by using polyclonal nonprimed CD4T cells in a clinically relevant allogeneic bone marrow transplantation (BMT) setting. We found that disruption of Th17-differentiation alone by targeting RORgammat (Th17-specific transcription factor) had no significant effect on GVHD development. We conclude that Th17 cells are sufficient but not necessary to induce GVHD.

Functional characterization and gene expression analysis of CD4+ CD25+ regulatory T cells generated in mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Although the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are mediated through binding and activation of the aryl hydrocarbon receptor (AhR), the subsequent biochemical and molecular changes that confer immune suppression are not well understood. Mice exposed to TCDD during an acute B6-into-B6D2F1 graft-vs-host response do not develop disease, and recently this has been shown to correlate with the generation of CD4(+) T cells that express CD25 and demonstrate in vitro suppressive function. The purpose of this study was to further characterize these CD4(+) cells (TCDD-CD4(+) cells) by comparing and contrasting them with both natural regulatory CD4(+) T cells (T-regs) and vehicle-treated cells. Cellular anergy, suppressive functions, and cytokine production were examined. We found that TCDD-CD4(+) cells actively proliferate in response to various stimuli but suppress IL-2 production and the proliferation of effector T cells. Like natural T-regs, TCDD-CD4(+) cells do not produce IL-2 and their suppressive function is contact dependent but abrogated by costimulation through glucocorticoid-induced TNFR (GITR). TCDD-CD4(+) cells also secrete significant amounts of IL-10 in response to both polyclonal and alloantigen stimuli. Several genes were significantly up-regulated in TCDD-CD4(+) cells including TGF-beta3, Blimp-1, and granzyme B, as well as genes associated with the IL12-Rb2 signaling pathway. TCDD-CD4(+) cells demonstrated an increased responsiveness to IL-12 as indicated by the phosphorylation levels of STAT4. Only 2% of TCDD-CD4(+) cells express Foxp3, suggesting that the AhR does not rely on Foxp3 for suppressive activity. The generation of CD4(+) cells with regulatory function mediated through activation of the AhR by TCDD may represent a novel pathway for the induction of T-regs.

Evaluation of topical ciclosporin 0.05% for prevention of rejection in high-risk corneal grafts.

AIM: To evaluate the efficacy of combined treatment with commercially available 0.05% topical ciclosporin and topical corticosteroid compared with treatment with topical corticosteroids only after high-risk keratoplasty. PATIENTS AND METHODS: A total of 47 high-risk keratoplasties were randomly divided into two groups based on the postoperative immunosuppression. Twenty-five eyes (group 1) were treated with 0.05% ciclosporin and dexamethasone 0.1%, and 22 eyes (group 2) were treated with dexamethasone only. The clinical outcome of penetrating keratoplasty was evaluated by the rate of rejection-free graft survival and graft survival evaluation by the Kaplan-Meier logrank test. RESULTS: The average length of follow-up was 20.2 (SD 7.1) months in group 1 and 18.5 (6.6) months in group 2 (p = 0.421). Rejection-free graft survival rates were 60.8% in group 1 and 54.5% in group 2 (Kaplan-Meier logrank test, p = 0.474). In group 1, the graft survival rate was 73.9%; in group 2, the graft survival rate was 68.1%. The difference in the graft survival rates between the groups was also not statistically significant (Kaplan-Meier logrank test, p = 0.518). CONCLUSION: In high-risk corneal grafts, the efficacy of 0.05 percent commercially available topical ciclosporin combined with dexamethasone topically was not better than that of dexamethasone alone in preventing rejection.

Chimerism studies as an approach for the induction of tolerance to extremity allografts.

SUMMARY: Recent advances in the field of transplant immunology and reconstructive surgery have resulted in an increased interest in extremity allograft. Until now, more than 20 hand transplants have been performed in humans. Rejection is well controlled by currently available immunosuppressive drugs. The hand transplant, however, is not a life-supporting organ transplant and these drugs are unlikely to represent the final solution for hand transplantation due to serious adverse effects. The ultimate goal of extremity allograft is the induction of donor-specific immunotolerance. The major strategies for tolerance induction are: (1) T-cell costimulation blockade, (2) induction of mixed chimerism, (3) T-cell depletion, and (4) tolerance mediated by regulatory T cells. Amongst these, the establishment of a high level of chimerism may be the most stable strategy for donor-specific tolerance, and our laboratory has been investigating the induction of macrochimerism following extremity allotransplantation. Recently, some studies demonstrated that macrochimerism induces immunotolerance for extremity allograft in the rodent model. We made a new protocol using cyclophosphamide (CYP) and granulocyte colony-stimulation factor (G-CSF) to induce high-level chimerism following rat whole-limb allotransplantation. Limb allografting could function as a vascularised carrier for bone marrow transplantation, providing a continuous source of donor cells and contributing to a high level of chimerism in the recipient. Pretransplant CYP followed by G-CSF and FK506 treatment significantly prolong the survival of limb allografts, but frequently cause chronic graft-versus-host disease in the recipients. In this review, recent experimental chimerism studies are presented for tolerance induction and we review the prospect of clinical applicability in extremity allograft.

After discontinuation of calcineurin inhibitors, tapering of mycophenolate mofetil further impairs donor-directed cytotoxicity.

BACKGROUND: Recently, we described a significant decrease in donor-specific cytotoxic T-lymphocyte precursor frequency (CTLpf) after discontinuation of calcineurin inhibitors (CNI), while the proliferative capacity in mixed lymphocyte culture (MLC), and the number of interferon-gamma (IFN-gamma) producing cells (pc) in Elispot remained unchanged. METHODS: We tested T-cell reactivity in CNI free patients with stable renal graft function, on mycophenolate mofetil (MMF) or azathioprine (AZA) plus prednisone, who were tapered to 50% of their MMF or AZA dose. RESULTS: Furthermore, tapering of the MMF or AZA dose resulted in a decrease of donor-reactive CTLpf in all patients with detectable CTLpf. Detectable numbers decreased from a median of 32 to 8 CTLp/10(6) peripheral blood mononuclear cell (PBMC). No effect on third-party reactive CTLpf was found, while the T-cell reactivity to donor and third-party cells as tested in MLC and in IFN-gamma Elispot was not affected either by tapering of immunosuppression. Third-party reactivity was significantly higher than donor-specific reactivity in all tests. A control group showed no changes in any of the in vitro assays. CONCLUSION: Both withdrawal of CNI and tapering of MMF or AZA dose decreases the donor-specific CTLpf. Our data suggest that reduction of immunosuppression results in a specific decrease of donor-directed cytotoxic capacity of immunocompetent cells, while their proliferation and cytokine production capacity remained unchanged. Immunosuppression hinders development of cytotoxic non-responsiveness.

The role of graft and host accommodation in a hamster-to-rat cardiac transplantation model.

BACKGROUND: We evaluated the importance and mechanism of graft and host accommodation in hamster-to-rat cardiac xenotransplantation models. METHODS: To evaluate graft accommodation, accommodated hamster grafts (Group 2) were transplanted to naïve host rats treated with FK506, and compared with naïve hamster grafts (Group 1). To evaluate host accommodation, three groups were evaluated: naive hamster hearts were transplanted to naïve hosts treated with FK506 (Group 3: 0.5 mg/kg, Group 4: 1.0 mg/kg) and splenectomy, and compared with accommodating hosts (Group 5) with FK506 0.5 mg/kg and splenectomy. We examined graft survival, histopathology, antihamster antibodies and B-1 cells in blood. RESULTS: Graft survival in Group 2 (3.4+/-0.9 days) was not significantly different from that in Group 1 (2.8+/-0.4 days). Graft survival in Groups 4 and 5 (>30 days) was significantly prolonged compared with that in Group 3 (6.0+/-0.7 days). Histopathology of Groups 1-3 showed humoral rejection, whereas Groups 4 and 5 showed normal histology and expression of protective genes. In Groups 1-3, antihamster immunoglobulin (Ig) M and B-1 cells increased significantly compared to Groups 4 and 5, where IgM and B-1 cells remained low or were reduced. CONCLUSIONS: Host accommodation was more important than graft accommodation. Accommodating grafts expressing protective genes were rejected with an elevation of both IgM and B-1 cells. In accommodated hosts, both IgM and B-1 cells decreased, suggesting that B-1 cells may be responsible for the production of antihamster antibodies. These results suggest that sufficient suppression of B-1 cells, resulting in decreased titers of antihamster antibodies, may play an important role in host accommodation.

Acute graft versus host disease.

Acute graft-versus-host disease (GVHD) occurs after allogeneic hematopoietic stem cell transplant and is a reaction of donor immune cells against host tissues. Activated donor T cells damage host epithelial cells after an inflammatory cascade that begins with the preparative regimen. About 35%-50% of hematopoietic stem cell transplant (HSCT) recipients will develop acute GVHD. The exact risk is dependent on the stem cell source, age of the patient, conditioning, and GVHD prophylaxis used. Given the number of transplants performed, we can expect about 5500 patients/year to develop acute GVHD. Patients can have involvement of three organs: skin (rash/dermatitis), liver (hepatitis/jaundice), and gastrointestinal tract (abdominal pain/diarrhea). One or more organs may be involved. GVHD is a clinical diagnosis that may be supported with appropriate biopsies. The reason to pursue a tissue biopsy is to help differentiate from other diagnoses which may mimic GVHD, such as viral infection (hepatitis, colitis) or drug reaction (causing skin rash). Acute GVHD is staged and graded (grade 0-IV) by the number and extent of organ involvement. Patients with grade III/IV acute GVHD tend to have a poor outcome. Generally the patient is treated by optimizing their immunosuppression and adding methylprednisolone. About 50% of patients will have a solid response to methylprednisolone. If patients progress after 3 days or are not improved after 7 days, they will get salvage (second-line) immunosuppressive therapy for which there is currently no standard-of-care. Well-organized clinical trials are imperative to better define second-line therapies for this disease. Additional management issues are attention to wound infections in skin GVHD and fluid/nutrition management in gastrointestinal GVHD. About 50% of patients with acute GVHD will eventually have manifestations of chronic GVHD.

Augmentation of cellular immunity by kigamicin D.

Kigamicin D did not show any immunosuppressive activity in mixed lymphocyte culture reaction and mitogen induced lymphocyte blastogenesis in vitro and graft versus host reaction in vivo. Natural killer cell activity in spleen cells was not affected by oral administration of kigamicin D. Instead, delayed-type hypersensitivity response to sheep red blood cells was stimulated at a broad dosage level. It is concluded that kigamicin D increases cellular immunity to specific antigen.

Differential sensitivity of T lymphocytes and hematopoietic precursor cells to photochemotherapy with 8-methoxypsoralen and ultraviolet A light.

The combination of 8-methoxypsoralen (8-MOP) and long wave ultraviolet radiation (UV-A) has immunomodulatory effects and might abolish both graft-vs-host and host-vs-graft reactions after allogeneic hematopoietic stem cell transplantation. In the present study, we have confirmed the sensitivity of T lymphocytes to 8-MOP treatment plus UV-A exposure as evidenced by the abrogation of the alloreactivity in mixed lymphocyte cultures as well as the inhibition of the response to phytohemagglutinin A. However, the clonogenic capacity of the bone marrow hematopoietic progenitors was inhibited with UV-A doses lower than the doses needed to inhibit T-lymphocytes alloreactivity. Moreover, long-term bone marrow cultures showed that 8-MOP plus UV-A treatment had detrimental effects on the more immature bone marrow stem cells. These data were confirmed when murine bone marrow graft was treated with 8-MOP, exposed to UV-A, then transplanted into semiallogeneic recipient mice. The treated cells could not maintain their clonogenic capacity in vivo resulting in death of all animals. Taken together, these data show that ex vivo 8-MOP plus UV-A treatment of the marrow graft cannot be used to prevent post-bone marrow transplantation alloreactivity.

Avian thymic hormone treatment of peripheral blood mononuclear cells from young chicks stimulates acute graft-versus-host reaction in chicken embryos.

Avian thymic hormone (ATH) is a parvalbumin produced by epithelial cells in the thymic cortex of chickens and circulates in the blood on a 5-day cycle. It stimulates precocious development of cell-mediated immunity. The effect of partially purified extracts of thymus (TE) and purified ATH were tested for their effect on the acute graft-versus-host reaction (GVHR). Treatment of chicks for their first 3-days of life did not enhance the acute GVHR produced by their PBMC in 14-day-old embryos. PBMC from 3-day-old chicks were treated in vitro with TE, ATH, thymosin fraction 5 or thymosin alpha1 for 2 h and injected into 14-day-old embryos. Bone marrow cells and thymic lymphocytes were treated with TE. Only PBMC treated with TE or ATH produced an enhanced acute GVHR. Because ATH targets gammadelta T cells, the data implicate participation of donor gammadelta T cells in the acute GVHR.

Haemato-biochemical and immuno-pathophysiological effects of chronic toxicity with synthetic pyrethroid, organophosphate and chlorinated pesticides in broiler chicks.

Haemato- biochemical and immuno-pathophysiological changes following feeding of broiler chicks with 20 ppm fenvalerate (synthetic pyrethroid, SP), 2 ppm monocrotophos (organophosphate, OP) and 2 ppm endosulfan (chlorinated hydrocarbon, CH) were studied. Four groups of broiler birds (30 each) were fed poultry mash without (control) or mixed with pesticides for 8 weeks. Blood glucose, serum globulin and acetyl cholinesterase (AChE) activity level were decreased (P<0.01) in all treated groups compared to control, but not the serum albumin and BUN. The total ATPase activity was enhanced (P<0.01) in fenvalerate and monocrotophos than birds in control group. Body weight, total erythrocyte count, packed cell volume, haemoglobin, eosinophil and monocyte count did not show any changes. Total leucocytes and T-lymphocyte count was lower (P<0.01) in all treated groups as compared to control group. B-cell count (P<0.01), mean 2-4-dinitrofluorobenzene (DNFB) dermal sensitivity score and splenic indices from graft vs. host reaction (P<0.05) were decreased in fenvalarate and endosulfan but the values for monocrotophos were intermediate between control and other treated groups. Pesticide intoxication reduced nitroblue tetrazolium (NBT) positive cells (active splenic macrophages) (P<0.05) and spleen weight (P<0.01). Whereas bursal weight was reduced only with endosulfan, thymic weight was reduced on endosulfan and fenvalerate-treated feed. Microscopic examination of these organs further revealed atrophy/hypoplasia, decrease in the size of follicles with depletion of lymphocytes and haemorrhages in thymus. The study concludes that the chronic exposure of chicks to small amount of SP, OP and CH pesticide leads to deleterious effects on metabolism and immune system of birds.