NetNews Usenet Archive 1993 #3/NN_1993_3.iso/spool/sci/med/aids/2276

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From: Billi Goldberg <[e-mail]>
Subject: CD8s, Clonal Anergy, and Fauci
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Note: Copyright 1992, Dan R. Greening. Non-commercial reproduction allowed.
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The following lead statement and the Go and Otten articles were faxed by
me on 1/23/93 to G. Pantaleo of Fauci's Lab.

What is very interesting is that amount of research being done by
Fauci's group on cytotoxic T cells. There are, of course, those that
will stick to CD4 counts until you know what freezes over, but the
cutting edge of research is on CTLs, NKs, and Th1/Th2, T1/T2 and how
they are influenced by cytokines especially lymphokines. The Fauci
articles are critical because they show a pattern and direction of
research.

Fauci's new article on the pathogenesis of AIDS should be published in
NEJM on 2/4/93. The delay was caused be required revisions and
shortening.
=======================================================================
          CD8 CELLS, CLONAL ANERGY AND FAUCI'S ARTICLES
                         by Billi Goldberg

I think that the Go et al. article helps explain some of the
proliferative problems of CD8+ HLA-DR+ in vitro. As I have stated
previously, when macrophages and LC/DC (professional antigen presenting
cells which saturate the mucosal tissues) are infected there appears to
be a costimulatory problem probably with CD28(T cell)/B7 (APC) which
could be either contact or signal transduction. This, then, could result
in a proliferative problem but wouldn't interfere with the lytic ability
of CD4+ (cytotoxic)  and CD8+. Langerhans cells differentiate into
veiled dendritic cells when migrating from the skin and then into
interdigitating dendritic cells in the lymph nodes; this is quite
different than follicular dendritic cells as you well know.

There seems to be little question about the HIV infection of LC/DC. It
is quite interesting that contact sensitization results in approximately
one-third of the cutaneous LC migrating from the skin to the nodes for
destruction. I have not seen any studies that mentions mucosal LC
migration, but it just might happen. These possibly infected cells would
be destroyed in the nodes and replaced by non-infected LC. By doing this
weekly, the co-stimulatory function of LC/DC would be preserved
eliminating the anergy that is probably the primary cause of the
decreased CD4s and CD8s during disease progression and inability to
control OIs. Interesting concept that just might explain why DNCB users
stay alive and control OIs. The Go et al. article explains how the APCs
could be destroyed by the anergized T cell clones.

It is too bad that the old-school immunologists have such a difficult
time accepting the professional antigen presenting capabilities of
dendritic cells. Too many immunologists just write them off as
specialized macrophages. Of course, that just may be so, but so what.
The only way to get systemic immune responses (takes about 5 days) in
the nodes is with dendritic cells; macrophages only result in localized
responses that last about 24 hours.

I think the Th1/Th2 and T1/T2 theories explain quite a bit. It is
interesting that CD8 suppressor cells might just be nothing more than a
lack of interferon-gamma and a surplus of IL-4. Maybe the secret might
be to shut down Th2/T2 which interferes with Th1/T1 by the production of
IL-10 that inhibits interferon-gamma production. Selgame et al. article
in Science, 10/11/91:289-282 is very interesting and might be well worth
reading again especially the part about CD8 cytotoxic and suppressor
cells. DNCB initiates a pure Th1 cell mediated immune response.
======================================================================
Go C, Lancki DW, Fitch FW, Miller J. Anergized T cell clones retain
their cytolytic ability. Journal of Immunology, January 15,
1993;150(2):367-376.

Abstract: CD4+ T cells have been described to have both helper and lytic
function. The helper function of Th1 cells in particular can be
inactivated by inducing the T cell into a state of nonresponsiveness in
which the T cell is no longer capable of producing IL-2 or proliferating
in an autocrine way to a conventional antigenic stimulus. To determine
whether the lytic ability of Th1 cells can also be rendered
nonfunctional upon anergy induction, we induced Th1 clones into a
nonresponsive state and tested their ability to lyse target cells in an
Ag-specific and MHC class II-restricted manner. We show that cells newly
induced into an anergic state were able to lyse target cells
nonspecifically. This effect was short-lived and after resting in
culture media, the cells regained their ability to lyse target cells in
an Ag/MHC-specific manner, and this ability was comparable to normal
resting T cells. In contrast, the helper function of these cells
remained non-responsive, and the cells were unable to proliferate or to
secrete IL-2 in response to the same antigenic stimulus used for lysis.
Therefore, the lytic pathway appears to be regulated separately from the
proliferative/lymphokine pathway(s) and is not affected long-term by an
anergic stimulus.

Page 373: The most striking observation from these studies is that
although anergized Th1 cells fail to secrete IL-2 and to proliferate to
restimulation, they retain normal lytic activity. Although there is a
slight decrease in Ag/MHC-specific cytolytic activity shortly after
induction of anergy, after long rest periods in culture, the anergized T
cells are able to lyse their target cells as specifically and
efficiently as resting T cells. The retention of lytic activity by these
cells suggest that activation of the IL-2 production pathway is
regulated separately from activation of the lytic pathway and that lytic
ability is not dependent on the presence of costimulation at the time of
TCR engagement. These results are in agreement with a recent analysis of
CD8-positive T cells, where IL-2 production, but not lytic ability, was
sensitive to anergy induction. In contrast, neither lymphokine
production nor lytic function of Th2 cells are sensitive to conditions
that induce anergy in Th1 and CD8 T cells clones.
======================================================================
Otten GR; Germain RN. Split anergy in a CD8+ T cell: receptor-dependent
cytolysis in the absence of interleukin-2 production. Science, 1991 Mar
8, 251(4998):1228-31.

Abstract: Engagement of the antigen-specific receptor (TCR) of CD4+
T lymphocytes without a second (costimulatory) signal prevents the
subsequent production of interleukin-2 (IL-2) by these cells. Because
IL-2 is a key immunoregulatory lymphokine and is also produced by a
subset of CD8+ T cells that are able to kill target cells, the effect of
engaging the TCR of one such clone in the absence of costimulatory
signals was examined. The capacity for TCR-dependent IL-2 production was
lost, indicating comparable costimulator-dependent signaling
requirements for IL-2 production in CD4+ and CD8+ T cells. However,
TCR-mediated cytotoxicity was not impaired, implying that costimulation
is required for only certain TCR-dependent effector functions.
======================================================================
De Maria A; Pantaleo G; Schnittman SM; Greenhouse JJ; Baseler M;
Orenstein JM; Fauci AS. Infection of CD8+ T lymphocytes with HIV.
Requirement for interaction with infected CD4+ cells and induction of
infectious virus from chronically infected CD8+ cells. Journal of
Immunology, 1991 Apr 1, 146(7):2220-6.

Abstract: In this study, we have investigated the basic requirements for
HIV-1 infection of CD8+ lymphocytes in vitro. Unfractionated PBL
obtained from healthy HIV-1 seronegative donors were activated with PHA
and infected in vitro with HIV-1LAV. Based on immunofluorescent
labeling, the vast majority of cells (85 to 97%) surviving peak virus
replication belonged to the CD8+ subset and only a small percentage (0.5
to 1.5%) were CD4+. Amplification of HIV-1 proviral sequences by
polymerase chain reaction performed on the sorted surviving CD8+ cells
demonstrated that CD8+ cells harbored HIV-1 proviral DNA. In addition,
stimulation of these HIV-1-infected, CD8(+)-sorted cells either with PHA
or anti-CD2 mAb resulted in induction of virus replication, as measured
by reverse transcriptase activity. Electron microscopic analysis of CD8+
cells chronically infected with HIV-1 and stimulated with PHA showed
typical virions budding from, and associated with, the surface of cells
immunolabeled with gold beads directed toward the CD8 molecule.
Infection of CD8+ cells with HIV-1 occurred only when CD4+ cells were
present in the PHA-activated lymphocyte population exposed to HIV-1 at
the beginning of the culture or when sorted CD8+CD4- lymphocytes were
cocultured with autologous sorted CD8-CD4+ cells that had been
previously infected with HIV-1. Coculture of these cells with PHA-blasts
and incubation of their supernatants with a CD4+ cell line showed that
these chronically infected CD8+ cells could spread HIV-1 infection to
uninfected CD4+ cells after stimulation with PHA or anti-CD2 mAb.
Therefore, these results suggest that the minimal requirement for in
vitro infection of CD3+CD8+CD4- lymphocytes is the presence of infected
CD4+ cells and that infected CD8+ T lymphocytes can further spread the
infection to CD4+ cells.
======================================================================
Pantaleo G; De Maria A; Koenig S; Butini L; Moss B; Baseler M; Lane HC;
Fauci AS. CD8+ T lymphocytes of patients with AIDS maintain normal broad
cytolytic function despite the loss of human immunodeficiency virus-
specific cytotoxicity. Proceedings of the National Academy of Sciences
of the United States of America, 1990 Jun, 87(12):4818-22.

Abstract: In this study, we have investigated the potential mechanisms
responsible for the loss of human immunodeficiency virus type 1
(HIV-1)-specific cytolytic activity in the advanced stages of HIV-1
infection. We have demonstrated that HIV-1-specific cytotoxic T
lymphocytes are predominantly contained within the CD8+DR+ subset.
Furthermore, we have shown by a redirected killing assay that there is a
dichotomy between HIV-1-specific cytolytic activity and broad cytolytic
potential since the cytolytic machinery of CD8+DR+ cells is still
functioning even in patients with AIDS who have lost their HIV-1-
specific cytolytic activity. In addition, by comparative analysis of
these two types of cytolytic activity over time we have demonstrated a
progressive loss of HIV-1-specific cytolytic activity in the advanced
stages of the disease, whereas the cytolytic potential remained
unchanged regardless of the clinical stage. As previously shown in
patients with AIDS, even in asymptomatic HIV-1-seropositive patients,
CD8+DR+ cells from the same patient, compared to CD8+DR- lymphocytes,
showed a substantial reduction in their ability to proliferate in vitro
in response to different stimuli, such as mitogens (phytohemagglutinin
and phorbol 12-myristate 13-acetate) and monoclonal antibodies directed
against CD3, CD2, and CD28 molecules, and displayed a defective
clonogenic potential. Thus, on the basis of these results we propose
that the loss of HIV-1-specific cytolytic activity in HIV-1-infected
individuals may result at least in part from a progressive decrease in
the pool of HIV-1-specific cytotoxic T lymphocytes belonging to the
CD8+DR+ subset whose ability to expand has been impaired.
======================================================================
Pantaleo G; Koenig S; Baseler M; Lane HC; Fauci AS. Defective clonogenic
potential of CD8+ T lymphocytes in patients with AIDS. Expansion in vivo
of a nonclonogenic CD3+CD8+DR+CD25- T cell population. Journal of
Immunology, 1990 Mar 1, 144(5):1696-704.

Abstract: This study examines the potential mechanism(s) responsible for
the defective clonability of CD8+ T lymphocytes in patients with AIDS.
By the combined use of one- and two-color fluorescence cytofluorometry
we have shown an increase in the number of circulating DR+ cells due to
the expression of DR on a relatively large proportion of T lymphocytes
(one-third of CD3+ cells), the majority of them belonging to the CD8+
subset. In addition, the majority of CD8+DR+ cells in AIDS patients did
not express CD25 Ag (the receptor for IL-2), a surface marker generally
expressed on normal activated T lymphocytes. Sorted CD8+DR+ and
CD8+DR- cell populations were analyzed comparatively for their ability
to proliferate in response to different stimuli, including anti-CD3,
anti-CD2, alone or in combination with anti-CD28 mAb and mitogens such
as PHA, alone or in combination with PMA. We have demonstrated that
CD8+DR+ cells were severely defective in their proliferative response to
triggering via these major pathways of T cell activation even when an
exogenous source of IL-2 or IL-4 was added to the microcultures 24 h
after initiating the cultures. In contrast, CD8+DR- cells showed a
significant proliferation in response to the different stimuli and the
proliferative response was strongly enhanced by the addition of IL-2 or
IL-4. At the end of the stimulation period CD8+DR+ and CD8+DR-
proliferating populations were analyzed for CD25 Ag expression. Only 1
to 10% of CD8+DR+ cells expressed CD25 antigen compared with 40 to 50%
of CD8+DR- cells. The proliferative defect of CD8+DR+ cells was further
confirmed in experiments performed at the clonal level. The analysis of
the frequency of proliferating T lymphocyte-precursors in both CD8+DR+
and CD8+DR- subsets showed that the defective clonogenic potential of
CD8+ cells in AIDS patients could be in large part ascribed to CD8+DR+
cells. Five percent of CD8+DR+ cells showed a clonogenic potential
compared to the 25% of CD8+DR- cells. Finally, we analyzed the surface
expression of VLA-2 Ag, a marker of a chronic state of T cell
activation, on circulating T lymphocytes. We have shown that a large
proportion of CD3+DR+CD25- cells (50 to 80% in the different patients
with AIDS analyzed) expressed VLA-2 Ag.(ABSTRACT TRUNCATED AT 400 WORDS)
======================================================================
Koenig S; Earl P; Powell D; Pantaleo G; Merli S; Moss B; Fauci AS.
Group-specific, major histocompatibility complex class I-restricted
cytotoxic responses to human immunodeficiency virus 1 (HIV-1) envelope
proteins by cloned peripheral blood T cells from an HIV-1-infected
individual. Proceedings of the National Academy of Sciences of the
United States of America, 1988 Nov, 85(22):8638-42.

Abstract: Freshly separated unfractionated peripheral blood mononuclear
cells (PBMC) and cloned cell lines from a healthy human immunodeficiency
virus 1 (HIV-1)-seropositive individual were examined for cytotoxic
responses to HIV proteins expressed by recombinant vaccinia viruses. It
was found that freshly isolated PBMC recognize variant envelope proteins
of HIV-1 but not a more distantly related envelope protein derived from
the simian immunodeficiency virus (SIVmac). Although the effector cells
were predominantly CD8+, both MHC-matched and -unmatched target cells
were lysed. Cytotoxic T lymphocyte (CTL) clones were found to lyse cells
expressing HIV-1 envelope or reverse transcriptase. In contrast to the
cytotoxic response detected with PBMC, the cloned CTLs were major
histocompatibility complex (MHC) class I restricted. Our finding that a
cloned CTL line lysed cells expressing highly divergent HIV envelopes
strongly suggested that a conserved epitope was recognized.
Identification of these shared epitopes may assist in designing a
vaccine for HIV-1 that could stimulate MHC-restricted cytotoxic
responses.