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Background: Progressive Multifocal Encephalopathy (PML) is a subacute infection of the central nervous system (CNS) mediated by John Cunningham (JC) polyomavirus (PyV). The disease is frequently fatal, unless adaptive immunity to JC virus is restored. The disease typically occurs in immunosuppressed patients (e.g. HIV/AIDS). In the modern era, patients with hematological malignancies treated with rituximab and/or fludarabine or following immunosuppressive therapy for transplant or autoimmunity (e.g. multiple sclerosis (MS) or Crohn's disease) are also at risk. JC-PML can also occur in patients with genetic defects of immunity. Specific treatments do not exist and immunocompromised patients with history of cancer or genetic defects of immunity have no realistic chance for rapid recovery of their ability to fight infections. In subjects with Rituximab-related PML the case-fatality ratio is >90%. Survivors can be left with severe neurological disabilities. Methods: Based on our previously established procedure for generation of multi-virus virus-specific T cells using overlapping peptide libraries (pepmixes) as immunogens, we have developed dedicated polyomavirus-specific T cells (PyVST) targeting polyomavirus BK large T (LT) and Viral Protein 1 (VP1) antigens and highly cross-reactive with the structurally-homologous JC LT and VP1 proteins (see Figure). We hypothesized that adoptive transfer of donor-derived PyVSTs could be safely used for therapy of patients with refractory JC-PML. We have developed a pilot study to test the feasibility and safety of adoptive immunotherapy strategy using PyVSTs from the partially matched healthy 1st degree relative donors (sibling, parent or offspring; NIH study 16-N-0072). Adults diagnosed with PML who have no other treatment options were eligible. Patients with MS or HIV were excluded. PyVSTs were generated from donor blood leukocytes cultured for 14 days in G-rex flasks upon stimulation with BK LT and VP1 pepmixes. Subjects underwent baseline physical examination, MRI and lumbar puncture. Upon enrollment subjects received an intravenous infusion of a fixed dose of 1x10e6 (+/-10%) PyVST cells/kg. Safety monitoring period was 28 days after each infusion. Subjects were eligible for up to two additional infusions (dose 2x10e6 PyVSTs/kg) a minimum of 28 days apart if no toxicities were observed. Serial MRI and lumbar punctures were performed to monitor response. Subjects were followed for 12 months after the last infusion. Results: Nine subjects have been enrolled and treated with at least one infusion of PyVSTs under this protocol and the trial is still accruing subjects. No immediate infusion reactions or adverse events have been observed within the safety monitoring period. No CNS immune system reconstitution syndrome (IRIS) has occurred. One subject received a single infusion and withdrew from the study following Day 14 visit due to unwillingness to travel. One subject received two doses of PyVSTs with stabilization of the disease, not requiring the 3rd infusion. This subject was withdrawn from the study just prior to the 1 year follow up visit, as he was diagnosed with the stage IV lung cancer and entered hospice. Seven subjects received 3 infusions of PyVSTs. 3 of these 7 patients died of refractory PML, all >30 days after the 3rd dose of T cells. At the time of this report one subject completed the study at one year with stable PML. The remaining subjects are in the follow-up phase of the protocol. Conclusions: We report that partially matched T cells targeting PyVs generated from healthy related donors can be safely used for adoptive immunotherapy of severely immunocompromised patients with PML. Multiple infusions of T cells are very well tolerated at the doses of up to 2x10e6 PyVST cells/kg, displaying excellent safety profile without adverse events. Furthermore, our data suggest possible efficacy of this strategy as a life-saving therapy for patients who otherwise face a dismal prognosis. Disclosures No relevant conflicts of interest to declare.

Recently, considerable interest has arisen as to use cord blood (CB) as a source of hematopoietic stem cells for allogenic transplantation when bone marrow (BM) from a familial HLA-matched donor is not available. Because human cytomegalovirus (HCMV) has been shown to inhibit the proliferation of BM progenitors in vitro, it was important to examine whether similar effect could be observed in HCMV-infected CB cells. Therefore, the effect of HCMV challenge on the proliferation of myeloid progenitors from BM and CB was compared using both mononuclear cells (MNC) and purified CD34+ cells. A clinical isolate of HCMV inhibited the colony formation of myeloid BM progenitors responsive to granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, macrophage-CSF, interleukin-3 (IL-3) and the combination of IL-3 and stem cell factor (SCF). In contrast, colony growth of CB progenitors was not affected. In addition, HCMV inhibited directly the growth of purified BM CD34+ cells responsive to IL-3 and SCF in single cell assay by 40%, wheras the growth of CD34+ progenitors obtained from CB was not suppressed. The HCMV lower matrix structural protein pp65 and HCMV DNA were detected in both CB and BM CD34+ cells after in vitro challenge. However, neither immediate early (IE)-mRNA nor IE proteins were observed in infected cells. Cell cyclus examination of BM and CB CD34+ cells revealed that 25.7% of BM progenitors were in S + G2/ M phase wheras only 10.7% of the CB progenitors. Thus, a clinical isolate of HCMV directly inhibited the proliferation of myeloid BM progenitors in vitro wheras CB progenitors were not affected. This difference in the susceptibility of CB and BM cells to HCMV may partly be caused by the slow cycling rate of naive CB progenitors compared to BM progenitors at the time of infection.

Bone marrow cells (BMC) are involved in the pathogenesis of human cytomegalovirus++ (HCMV) infections, and the hematopoietic cells are probable sites of HCMV latency in healthy donors. In vitro studies have indicated both a direct inhibitory effect of HCMV on proliferation and differentiation of myeloid bone marrow progenitors and an impairment of bone marrow stroma cell function by HCMV. The purpose of the present study was to establish whether the suppressing effect could be limited to subsets of immature CD34+ BMC and to investigate the role of immature cell populations as possible sites of HCMV latency. CD34+ cells from healthy HCMV-seropositive and -seronegative donors were sorted according to the expression of HLA-DR (CD34+ HLA-DR+ and CD34+ HLA-DR- cells). The progenitor growth of hematopoietic progenitor cells from seronegative donors was examined by colony and single-cell assays after in vitro infection with HCMV. To determine the susceptibility of the CD34+ cells to HCMV infection in vitro and in vivo, cells of both subsets from seronegative and seropositive donors were analyzed for the presence of HCMV DNA by polymerase chain reaction. HCMV infection in vitro inhibited the interleukin-1alpha (IL-1alpha)-, IL-3-, granulocyte colony-stimulating factor-, granulocyte-macrophage colony-stimulating factor-, and stem cell factor-induced proliferation in single-cell assays of CD34+ HLA-DR-cells by 34%. In contrast, the colony growth of the CD34+ HLA-DR+ subset was suppressed in cells from only 3 of the 8 donors. However, in vitro HCMV infection of the CD34+ HLA-DR+ progenitor cells inhibited the proliferation of all donors tested when hematopoietic growth factors were used individually to promote progenitor growth. In addition, the formation of burst-forming units-erythroid and colony-forming units-granulocyte, erythrocyte, monocyte, megakaryocyte was reduced 40% to 60% by HCMV in vitro. In contrast, the growth of high proliferative potential colony-forming cells was not inhibited after in vitro HCMV infection. Furthermore, HCMV DNA was detected in both CD34+ HLA-DR- and CD34+ HLA-DR+ progenitors from in vitro-infected HCMV-seronegative donors and cells from HCMV- seropositive donors. Taken together, the early progenitors defined as CD34+ HLA-DR- and CD34+ HLA-DR+ are directly suppressed in their proliferation by HCMV in vitro, and hematopoietic stem cells are also sites of HCMV latency in healthy HCMV-seropositive donors.

Recently, considerable interest has arisen as to use cord blood (CB) as a source of hematopoietic stem cells for allogenic transplantation when bone marrow (BM) from a familial HLA-matched donor is not available. Because human cytomegalovirus (HCMV) has been shown to inhibit the proliferation of BM progenitors in vitro, it was important to examine whether similar effect could be observed in HCMV-infected CB cells. Therefore, the effect of HCMV challenge on the proliferation of myeloid progenitors from BM and CB was compared using both mononuclear cells (MNC) and purified CD34+ cells. A clinical isolate of HCMV inhibited the colony formation of myeloid BM progenitors responsive to granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, macrophage-CSF, interleukin-3 (IL-3) and the combination of IL-3 and stem cell factor (SCF). In contrast, colony growth of CB progenitors was not affected. In addition, HCMV inhibited directly the growth of purified BM CD34+ cells responsive to IL-3 and SCF in single cell assay by 40%, wheras the growth of CD34+ progenitors obtained from CB was not suppressed. The HCMV lower matrix structural protein pp65 and HCMV DNA were detected in both CB and BM CD34+ cells after in vitro challenge. However, neither immediate early (IE)-mRNA nor IE proteins were observed in infected cells. Cell cyclus examination of BM and CB CD34+ cells revealed that 25.7% of BM progenitors were in S + G2/ M phase wheras only 10.7% of the CB progenitors. Thus, a clinical isolate of HCMV directly inhibited the proliferation of myeloid BM progenitors in vitro wheras CB progenitors were not affected. This difference in the susceptibility of CB and BM cells to HCMV may partly be caused by the slow cycling rate of naive CB progenitors compared to BM progenitors at the time of infection.

Calf kidney has been used as a tissue source for the isolation of cobalamin analogues, which are defined here as cobalt-containing compounds of distinctive chromatographic behavior that are extractable from tissues by methods conventionally used to extract cobalamin and which, in radioisotope dilution assays, are more active with R-protein as binder than intrinsic factor and are relatively less active in microbiologic assays. Preparatory methods employed reverse affinity chromatography or a series of chemical extractions for the isolation of corrin followed by Dowex-50 chromatography. An analogue-containing fraction (peak 2) was eluted by acetate buffers between pH 4 and 5. This material was shown to contain cobalt, to migrate differently than the four cobalamins in Dowex-50 and paper chromatography, and to display a pattern of properties that is compatible with the above definition of cobalamin analogues. These analogues stimulated crude preparations of N5-methyltetrahydrofolate-homocysteine methyltransferase (EC 2.1.1.13) from Escherichia coli and rat liver at far lower concentrations (1–40 nmol/L) than the major cobalamins. No evidence of enzymatic conversion of cobalamin to analogue could be demonstrated.

Data are presented correlating 22 determinations of the histamine content of blood in patients with chronic granulocytic leukemia with (1) the basophils (2) the other myeloid granulocytes. Expressing the histamine in each case on the basis of the amount in 108 granulocytes, there is a readily evident positive correlation with the basophil percentage and a negative correlation with the percentage of other myeloid elements. This is strikingly apparent in instances where blood with a very high percentage of basophils was analyzed. The data indicate that in chronic granulocytic leukemia, the basophil is predominantly and, perhaps exclusively, responsible for the marked elevations in blood histamine. Inferentially, it appears probable that this cell type, though small in numbers, may be the principal carrier of histamine in non-leukemic blood.

Studies are presented on the dextran, fibrinogen, and phytohemagglutinin methods of leukocyte separation. Optimal conditions, obtainable yields, detailed directions, advantages and disadvantages of each procedure are presented. It is shown that by using these methods in combinations, leukocyte suspensions of extreme purity may be obtained.

Calf kidney has been used as a tissue source for the isolation of cobalamin analogues, which are defined here as cobalt-containing compounds of distinctive chromatographic behavior that are extractable from tissues by methods conventionally used to extract cobalamin and which, in radioisotope dilution assays, are more active with R-protein as binder than intrinsic factor and are relatively less active in microbiologic assays. Preparatory methods employed reverse affinity chromatography or a series of chemical extractions for the isolation of corrin followed by Dowex-50 chromatography. An analogue-containing fraction (peak 2) was eluted by acetate buffers between pH 4 and 5. This material was shown to contain cobalt, to migrate differently than the four cobalamins in Dowex-50 and paper chromatography, and to display a pattern of properties that is compatible with the above definition of cobalamin analogues. These analogues stimulated crude preparations of N5-methyltetrahydrofolate-homocysteine methyltransferase (EC 2.1.1.13) from Escherichia coli and rat liver at far lower concentrations (1-40 nmol/L) than the major cobalamins. No evidence of enzymatic conversion of cobalamin to analogue could be demonstrated.